Compositions and methods for increasing animal size and growth rate

ABSTRACT

The present invention relates to germ line and somatic cells comprising a mutant p27 kip1  protein lacking a Cdk2 phosphorylation site. Also provided are transgenic animals and methods of making such transgenic animals which have increased size and/or growth rate.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/352,391, filed on Jan. 28, 2003, the disclosure of which isincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported by a grant from the National Institutes ofHealth (Grant No. CA-67893). The government may have certain rights inthe invention.

BACKGROUND OF THE INVENTION

Animal cells have both a proliferating phase and a quiescent phase.Cells can shift from the proliferating phase to the quiescent phaseduring a brief window in the cell cycle. Depending on their position inthe cell cycle, cells deprived of mitogens such as those present inserum can undergo immediate cell cycle arrest, or they can complete thecurrent mitotic cycle and arrest in the next cell cycle. The transitionfrom mitogen-dependence to mitogen-independence occurs in mid- tolate-G1 phase of the cell cycle. Anti-mitogenic signals can cause thecell cycle to arrest at a kinetically common point. In particular, inearly G1, cells can exit the cell cycle. Cell cycle commitment (autonomyfrom mitogenic signals) occurs in mid-G1.

The transition of cells through G1 and entry into S phase requires theaction of cyclin-dependent kinases (Cdks). Growth inhibitory signalshave been shown to prevent activation of these Cdks during G1 (Serranoet al., Nature 366:704-07 (1993); Hannon and Beach, Nature 371:257-61(1994); Xiong et al., Nature 366:701-04 (1993); Polyak et al., Cell78:59-66 (1994); Lee et al., Genes &Development 9:639-49 (1995); Koff etal., Science 260:536-39 (1993)). The catalytic activity of Cdks is knownto be regulated by two general mechanisms: protein phosphorylation andassociation with regulatory subunits (Gould et al., EMBO J. 10:3297-309(1991); Solomon et al., EMBO J. 12:3133-42 (1993); Solomon et al., Mol.Biol. Cell 3:13-27 (1992); Jeffrey et al., Nature 376:313-20 (1995);Morgan, Nature 374:131-34 (1995)). Among the regulatory subunits, theassociation of Cdks with inhibitory CKI subunits (Cyclin-dependentKinase Inhibitors) has been most closely correlated with the effect ofmitogen depletion on cell proliferation and Cdk activity.

The CKI directly implicated in mitogen-dependent Cdk regulation isp27^(Kip1) (Polyak et al., Cell 78:59-66 (1994); Toyoshima and Hunter,Cell 78:67-77 (1994)). Wildtype p27^(Kip1) protein accumulates to highlevels in quiescent cells, and is rapidly destroyed after quiescentcells are re-stimulated with specific mitogens (Nourse et al., Nature372:570-73 (1994); Kato et al., Cell 79:487-96 (1994)). The destructionof p27^(Kip1) is controlled by phosphorylation of p27^(Kip1) atthreonine 187 (T187). T187 is phosphorylated by Cdk2 to create a bindingsite for a Skp2-containing ubiquitin-protein ligase known as theSkp1-cullin-F-box protein ligase (SCF) (Feldman et al., Cell 91:221-30(1997); Bai et al., Cell 86:263-74 (1996); Skowyra et al., Cell91:209-19 (1997)). Ubiquitination of p27^(Kip1) by the SCF then resultsin p27^(Kip1) degradation by the proteosome (Sutterluty et al., NatureCell Biol. 1:207-14 (1999); Rolfe et al., J. Mol. Med. 75:5-17 (1997);Carrano et al., Nature Cell Biol. 1:193-99 (1999); Tsvetkov et al.,Curr. Biol. 9:661-64 (1999)).

The destruction of p27^(Kip1) was thought to be required for entry intoS phase. Moreover, constitutive expression of p27^(Kip1) in culturedcells causes the cell cycle to arrest in G1 (Polyak, supra; Toyoshimaand Hunter, supra). Thus, based on these observations, it was expectedthat cells harboring a null allele of p27^(Kip1) would arrest G1. It wassurprising, therefore, that animals harboring a null allele of thep27^(Kip1) gene survived. Indeed, such animals were larger than normal(increased animal size) and without apparent gross morphologicabnormalities. (Fero et al., Cell 85:733-44 (1996); U.S. Pat. No.5,958,769; the disclosures of which are incorporated by referenceherein.) The advantages of producing larger animals are readilyapparent, and include increase meat, milk and/or egg production.

Decreased levels of p27^(Kip1) in animals, however, cause certain minordefects, such as an ovulatory defect, and resulting female sterility,increased pituitary tumorigenesis and disrupted retinal architecture.(Fero et al., supra.) These defects can interfere with some uses of suchanimals. Thus, there is a need for alternative mutant alleles ofp27^(Kip1), and of methods of using such mutant alleles, that promoteincreased animal size or growth rate without these side effects.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acids encoding a mutantp27^(Kip1) protein that lacks a Cdk2 phosphorylation site, and to cellsharboring mutant p27^(Kip1) genes. In related aspects, transgenic cellsand transgenic animals are provided that have one or more mutantp27^(Kip1) genes encoding protein that lacks a Cdk2 phosphorylationsite.

In one aspect, isolated transgenic cells are provided comprising amutant p27^(Kip1) gene lacking a Cdk2 phosphorylation site. The mutantp27^(Kip1) gene encodes a mutant p27^(Kip1) protein having a longerhalf-life in S phase than wildtype p27^(Kip1) polypeptide. In certainembodiments, the mutant p27^(Kip1) polypeptide can inhibit Cdk2 in vitrokinase activity. In an embodiment, the mutant p27^(Kip1) polypeptide isp27^(T187A).

The mutant p27^(Kip1) gene can be located, for example, at an endogenousp27^(Kip1) locus; the endogenous locus can be heterozygous or homozygousfor the mutant p27^(Kip1) gene. The transgenic cell can be, for example,a primordial germ cell, oocyte, egg, spermatocyte, sperm cell,fertilized egg, zygote, embryonic stem cell, or somatic cell. Thetransgenic cell can also be progeny of any of these.

In another aspect, non-human, transgenic animals are provided whichcomprise a nucleic acid sequence encoding a mutant p27^(Kip1) proteinlacking a Cdk2 phosphorylation site. In an embodiment, the mutantp27^(Kip1) protein is p27^(T187A). The transgenic animal can be, forexample, a primate, mammal, bovine, porcine, ovine, equine, avian,rodent, fowl, piscine, or crustacean. In certain embodiments, thetransgenic animal is a farm animal, such as a chicken, cow, bull, horse,pig, sheep, goose or duck.

In a related aspect, a transgenic, non-human animal is provided whosegenome comprises a p27^(Kip1) gene and expresses a mutant p27^(Kip1)polypeptide having a longer half-life in S phase than wildtype p27polypeptide. Expression of the mutant p27^(Kip1) polypeptide results inincreased size or growth rate of the animal. The transgenic animal, canbe, for example, a primate, mammal, bovine, porcine, ovine, equine,avian, rodent, fowl, piscine, or crustacean. In certain embodiments, thetransgenic animal is a farm animal, such as a chicken, cow, bull, horse,pig, sheep, goose or duck.

Methods of increasing the size or growth rate of a non-human, transgenicanimal are also provided. Such methods generally include stablyintroducing into a genome of an animal cell a mutant p27^(Kip1) genelacking a Cdk2 phosphorylation site; and producing an animal from theanimal cell. In an embodiment, the method further includes transferringa nucleus from the animal cell into a second cell from which an animalcan be reconstituted; and allowing the second cell to develop into animmature animal. The immature animal typically is larger than animmature animal not having the mutant p27^(Kip1) gene. The second cell,can be, for example, an enucleated fertilized egg.

In another embodiment, the mutant p27^(Kip1) gene can be homologouslyintegrated at an endogenous p27^(Kip1) locus in the animal cell. Themutant p27^(Kip1) gene can be homologous or heterologous to the animalcell, and can be integrated at an endogenous p27^(Kip1) locus or at anon-p27^(Kip1) locus. The mutant p27^(Kip1) gene can encode, forexample, p27^(T187A) protein.

The animal cell can be, for example, a germ cell, a totipotent cell, astem cell, an embryonic stem cell, a pluripotent stem cell, a fetalcell, a primordial germ cell, an oocyte, an egg, a spermatocyte, a spermcell, a fertilized egg, a zygote, a blastomere, or a somatic cell. Theanimal cell can be a vertebrate cell, such as, for example, from aprimate, mammal, bovine, porcine, ovine, equine, avian, rodent, fowl,piscine, or crustacean. Exemplary animals include a chicken, hen,rooster, cow, bull, duck or goose.

Mutant genes can be introduced into cells by electroporation,microinjection, lipofection, transfection, biolistics, and the like. Themutant p27^(Kip1) genes can be introduced alone or as part of anexpression cassette that includes, for example, a heterologous promoteroperably associated with an open reading frame encoding a mutantp27^(Kip1) gene operably associated with a polyadenylation sequence. Theexpression cassette can also optionally include a selectable marker,such as the neomycin resistance gene. In an embodiment, the expressioncassette can be introduced into a cell using a viral vector.

In another aspect, a method for making a large fowl is provided. Themethod includes introducing a mutant p27^(Kip1) gene lacking a Cdk2phosphorylation site into the genome of a fowl cell by contacting invivo a blastodermal cell of a fertilized cell with the mutant p27^(Kip1)gene, wherein the p27^(Kip1) gene is introduced directly into thegerminal disk of the egg. Suitable fowl cells include those fromchickens, ostriches, emus, turkeys, ducks, geese, quail, parrots,parakeets, cockatoos or cockatiels.

A further understanding of the present invention will be obtained byreference to the following description that sets forth illustrativeembodiments.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention relates to nucleic acids encoding a mutantp27^(Kip1) protein that lacks a Cdk2 phosphorylation site and to cellsharboring mutant p27^(Kip1) genes. In related aspects, transgenic cellsand transgenic animals are provided that have one or more mutantp27^(Kip1) genes encoding protein that lacks a Cdk2 phosphorylationsite.

The p27^(Kip1) protein is phosphorylated by a Cdk at a phosphorylationsite to create a recognition sequence for a SCF (e.g., Cdk2). Theabsence or alteration of the Cdk2 phosphorylation site in p27^(Kip1)reduces or eliminates phosphorylation. Although the mutant p27^(Kip1)polypeptide is degraded in mid-G1 by the same pathway that degradeswildtype p27, the mutant p27^(Kip1) polypeptide has a longer half lifein the S phase of the cell cycle as compared with wildtype p27^(Kip1)polypeptide. Yet, the mutant p27^(Kip1) protein can retain otherfunctions, such as the ability to inhibit Cdk2 in vitro kinase activity.

In one aspect of the invention, isolated mutant p27^(Kip1) genes areprovided for introduction into animal cells. (The term “isolated” refersto a molecule, such as a nucleic acid, or cell, that has been removedfrom its natural cellular environment. For example, an isolated nucleicacid is typically at least partially purified from other cellularnucleic acids, polypeptides and other constituents.) The mutantp27^(Kip1) gene encodes a p27^(Kip1) polypeptide lacking a Cdk2phosphorylation site, such that less than about 10% of the mutantp27^(Kip1) polypeptide is phosphorylated at the Cdk2 phosphorylationsite. In certain embodiments, phosphorylation at the Cdk2phosphorylation site is less than about 5%, or less than about 1%.

The Cdk2 phosphorylation site can be defined by the following four aminoacid consensus sequence: (Ser/Thr)ProXaa(Lys/Arg) or the consensussequence (Ser/Thr)ProXaa(Lys/Arg/His/Pro), wherein Xaa can be any aminoacid residue. (See, e.g., Holmes and Solomon, J. Biol. Chem.271:25240-46 (1996).) Phosphorylation can be inhibited by substitutions,insertions and/or deletions (e.g., 1-3 amino acid insertions ordeletions).

Referring to Table 1, the Cdk2 phosphorylation site, including thephosphorylated residue, is generally conserved in p27^(Kip1)polypeptides. As shown in the table, an asterisk indicates the positionof a conserved threonine at position 187 of the human Cdk2phosphorylation site. As used herein, this conserved threonine isreferred to as threonine 187 (T187), although the skilled artisan willappreciate that this conserved residue may not be at position 187 in allp27^(Kip1) polypeptides. For example, in the mouse, hamster and ratpolypeptide sequences, the conserved, phosphorylated residue is atposition 186, although it is identifiable by sequence alignment and bybiochemical analysis, as discussed in the Examples (infra). Thus, theterms “T187,” “T187A” and position “187” are merely TABLE 1                                         * Consensus 151IRKRPATDDSSTQNKRANRTEENVSDGSPNAGSVEQTPKKPGLRRRQT 198 (SEQ ID NO:4)Genbank Species Residues  7769665 Human 151........................L....................... 198 SEQ ID NO:5) 4757962 Human 151 ................................................ 198SEQ ID NO:6) 12805035 Human 151................................................ 198 SEQ ID NO:6) 2135228 Human 151 ................................................ 198SEQ ID NO:6)  3913222 Cat 151...........P.................................... 198 SEQ ID NO:7)13429931 Pig 151 ...........P..................SA................ 198SEQ ID NO:8)  6753386 Mouse 151M.....AE...S....................T............Q   196 SEQ ID NO:9) 2493565 Hamster 151 M.....A....S................L................H..198 SEQ ID NO:10)  2102649 Rat 151M.....AE...S....................T............Q   196 SEQ ID NO:11) 2281010 Rat 151 M.....AE...S.....S..............T............Q   196SEQ ID NO:12)shorthand for this conserved threonine residue position and not to belimited to amino acid 187 of a p27^(Kip1) polypeptide, or thecorresponding codon in a p27^(Kip1) gene.

A Cdk2 phosphorylation site in a p27^(Kip1) polypeptide can beidentified, for example, by biochemical analysis. (See, e.g., Holmes andSolomon, supra.) A Cdk2 phosphorylation site in a p27^(Kip1) gene and/orpolypeptide sequence also can be identified by alignment with knownp27^(Kip1) gene and/or polypeptide sequences. For example, an alignmentcan be performed by the local homology algorithm of Smith and Waterman(Adv. Appl. Math. 2:482 (1981), which is incorporated by referenceherein in its entirety), by the homology alignment algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48:443-53 (1970), which isincorporated by reference herein in its entirety), by the search forsimilarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA85:2444-48 (1988), which is incorporated by reference herein in itsentirety), by computerized implementations of these algorithms (e.g.,GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage (Accelrys), or by visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show the percent sequence identity. It also plotsa tree or dendogram showing the clustering relationships used to createthe alignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which isincorporated by reference herein in its entirety). The method used issimilar to the method described by Higgins and Sharp (Comput. Appl.Biosci. 5:151-53 (1989), which is incorporated by reference herein inits entirety). The program can align up to 300 sequences, each of amaximum length of 5,000 nucleotides or amino acids. The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program is run by designating specific sequences andtheir amino acid or nucleotide coordinates for regions of sequencecomparison and by designating the program parameters. For example, areference sequence can be compared to other test sequences to determinethe percent sequence identity relationship using the followingparameters: default gap weight (3.00), default gap length weight (0.10),and weighted end gaps.

Another example of an algorithm that is suitable for aligning sequences,and for determining percent sequence identity and sequence similarity,is the BLAST algorithm, which is described by Altschul et al. (J. Mol.Biol. 215:403-410 (1990), which is incorporated by reference herein inits entirety). (See also Zhang et al., Nucleic Acid Res. 26:3986-90(1998; Altschul, et al., Nucleic Acid Res. 25:3389-402 (1997), which areincorporated by reference herein in their entirety). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction is halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefault parameters a wordlength (W) of 11, the BLOSUM62 scoring matrix(see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19(1992), which is incorporated by reference herein in its entirety)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The skilled artisan will appreciate, however, thatother parameters can be used.

The isolated, mutant p27^(Kip1) genes can be, for example, genomic DNA,cDNA, RNA, mRNA, and the like, as well as fragments of any of these. Themutant p27^(Kip1) genes can be polynucleotides or nucleic acids or otherpolymers composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds. Mutant genes can be of substantially any length,typically from about twelve (12) nucleotides to about 10⁹ nucleotides orlarger, that do not encode a Cdk2 phosphorylation site. In oneembodiment, a fragment of a mutant p27^(Kip1) gene has at least 50contiguous nucleotides; in other embodiments, the fragment of the mutantp27^(Kip1) gene is at least 100 nucleotides, at least 200 nucleotides,at least 500 nucleotides, at least 1000 nucleotides, or more of thegene. In related embodiments, the mutant p27^(Kip1) gene is at least anexon, a cDNA, or a fall length genomic p27^(Kip1) gene, lacking a Cdk2phosphorylation site.

Mutant p27^(Kip1) genes also include derivatives, such as those based onall possible codon choices for an amino acid(s) that, when expressedfrom a mutant p27^(Kip1) gene, results in the expression of a mutantprotein in which Cdk-mediated phosphorylation is inhibited. At aminoacid positions outside the Cdk2 phosphorylation site, mutant p27^(Kip1)gene derivatives can include those based on all possible codon choicesfor the same amino acid and codon choices based on conservative aminoacid substitutions. For example, the following six groups each containamino acids that are conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton,Proteins, W. H. Freeman and Company (1984).) In addition, individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids in an encoded sequenceare also “conservative substitutions.”

In certain embodiments, mutant p27^(Kip1) genes be synthesized, orchemically or biochemically modified (e.g., can contain non-natural orderivatized nucleotide bases). Such modifications include, for example,labels, methylation, substitutions of one or more of thenaturally-occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, and the like), chargedlinkages (e.g. phosphorothioates, phosphorodithioates, and the like),pendent moieties (e.g., polypeptides), intercalators (e.g., acridine,psoralen, and the like), chelators, alkylators, and modified linkages(e.g., alpha anomeric nucleic acids, and the like).

The mutant p27^(Kip1) gene(s) can be homologous or heterologous to thecell or the animal. As used herein, the term “homologous” p27^(Kip1)gene refers to a p27^(Kip1) gene derived from the same species as thecell or animal. A “heterologous” p27^(Kip1) gene refers to a p27^(Kip1)gene from a different species. For example, if the animal is a chicken,a homologous mutant p27^(Kip1) gene is derived from a chicken p27^(kip1)gene, while a heterologous mutant p27^(Kip1) gene is derived, forexample, from a mouse p27^(Kip1) gene.

The mutant p27^(Kip1) gene can be prepared by, for example, mutagenizinga wild-type p27^(Kip1) gene at one or more positions in the Cdk2phosphorylation site. In various embodiments, the p27^(Kip1) gene ishuman, primate, mammalian, avian, porcine, ovine, bovine, fowl, rodent,fish, crustacean, and the like. In specific embodiments, the p27^(Kip1)is from a sheep, goat, horse, cow, bull, pig, rabbit, guinea pig,hamster, rat, gerbil, mouse, chicken, ostrich, emu, turkey, duck, goose,quail, parrot, parakeet, cockatoo, cockatiel, trout, cod, salmon, crab,king crab, lobster, shrimp, and the like. p27^(Kip1) gene sequences aredisclosed for example, in the GenBank database under accession numbersgi|7769665|, gi|4757962|, gi|12805035|, gi|2135228|, gi|3913222|,gi|13429931|, gi|6753386|, gi|2493565|, gi|2102649|, and gi|2281010|,which are incorporated by reference herein in their entirety. p27^(Kip1)polypeptide sequences are disclosed, for example, in the GenBankdatabase under accession numbers AAF69497.1, NP_(—)004055.1, AAH01971.1,I52718, O19001, BAB39725.1, NP_(—)034005.1, Q60439, BAA19960.1, andBAA21561.1 (the disclosures of which are incorporated by referenceherein in their entirety).

p27^(Kip1) genes can be readily isolated by methods known to the skilledartisan. (See generally Sambrook et al., Molecular Cloning, A LaboratoryManual, 3rd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y.(2001); Ausubel et al., Current Protocols in Molecular Biology, 4th ed.,John Wiley and Sons, New York (1999); which are incorporated byreference herein in their entirety.) Specific embodiments for theisolation of p27^(Kip1) genes, presented as example but not by way oflimitation, are described below.

p27^(Kip1) genes can be isolated, for example, by polymerase chainreaction (PCR) to amplify the p27^(Kip1) gene, or a portion thereof,from a genomic or cDNA library. Oligonucleotide primers representingknown p27^(Kip1) sequences can be used as primers in PCR. In a typicalembodiment, the oligonucleotide primers represent at least a fragment ofconserved segments of identity between p27^(Kip1) genes of differentspecies. Synthetic oligonucleotides can be utilized as primers toamplify particular oligonucleotides within a p27^(Kip1) gene by PCRsequences from any suitable source (e.g., RNA or DNA), typically a cDNAlibrary or mRNA of potential interest. PCR can be carried out, forexample, by use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (Gene Amp). Degenerate primers can be designed for use in thePCR reactions. For example, the CODEHOP strategy of Rose et al. (Nucl.Acids Res. 26:1628-35 (1998), which is incorporated by reference hereinin its entirety) can be used to design degenerate PCR primers usingmultiply-aligned sequences as a reference. Methods for performing PCRand related methods are well known in the art. (See, e.g., U.S. Pat.Nos. 4,683,202; 4,683,195 and 4,800,159; Innis et al., PCR Protocols: AGuide to Methods and Applications, Academic Press, Inc., San Diego,Calif. (1989); Innis et al., PCR Applications: Protocols for FunctionalGenomics, Academic Press, Inc., San Diego, Calif. (1999); White (ed.),PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering,Humana Press, (1996); EP 320 308; the disclosures of which areincorporated by reference herein in their entirety.)

In an embodiment, degenerate primers are used to isolate the p27^(Kip1)cDNA from an avian species. Avian species are known to have p27^(Kip1).(See Torchinsky et al., J. Neurocytol. 28:913-24 (1999).) Briefly, analignment of multiple p27^(Kip1) polypeptide sequences from differentanimals is prepared and used to visually identify blocks of sequenceshaving low codon degeneracy (see Rose et al. (supra)). The CODEHOPstrategy of Rose et al. (supra) is used to design degenerate primersbased on the blocks of low codon degeneracy. Pools of primers varying inredundancy from 2 fold to about 32 fold are prepared. A hemi-nested PCRstrategy is used to amplify fragments from an avian chicken cDNA library(e.g., a chicken or hyacinth macaw library from Stratagene). Briefly,PCR is performed at 55° C. using the primer pools. (See, e.g., Rose etal. (supra); Rose et al., J. Virology 71:4138-44 (1997).) PCRamplification products can be detected, for example, by agarose gelelectrophoresis. The identity of the PCR amplification products can beconfirmed by DNA sequence analysis. Once the identify of the PCRamplification products is confirmed, the amplification products can beused to isolate full length p27^(Kip1) cDNA from the avian cDNA library.(See, e.g., Sambrook et al., supra; Ausubel et al., supra.)

For expression cloning (a technique commonly known in the art), anexpression library is constructed by methods known in the art. Forexample, mRNA is isolated, cDNA is prepared and then ligated into anexpression vector (e.g., a bacteriophage derivative) such that it iscapable of being expressed by the host cell into which it is thenintroduced. Various screening assays can then be used to select for theexpressed p27^(Kip1) polypeptide. In one embodiment, polyclonalantibodies against a mammalian p27^(Kip1) polypeptide (see, e.g. U.S.Pat. No. 6,242,575; the disclosure of which is incorporated by referenceherein in its entirety) are used to screen a chicken cDNA expressionlibrary (e.g., from Strategene) to identify avian p27^(Kip1) genes.

Alternatively, p27^(Kip1) genes can be isolated by hybridization using aheterologous p27^(Kip1) nucleic acid as a probe. For example, p27^(Kip1)genes can be isolated by screening a genomic or cDNA library with ap27^(Kip1) nucleic acid probe. Such a probe can be, for example, aportion of a p27^(Kip1) gene or its specific RNA, or a fragment thereof,that exhibits low codon degeneracy. Such a probe can be prepared,detectably labeled, and used to screen a library by nucleic acidhybridization (see, e.g., Benton and Davis, Science 196:180-82 (1977);Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72:3961-65 (1975);Sambrook et al., supra; Ausubel et al., supra). DNA fragments withsubstantial identity to the probe will hybridize and can be identifiedusing the detectable label.

In various embodiments, hybridization screening using a heterologousp27^(Kip1) nucleic acid probe can assist in the isolation of p27^(Kip1)genes. p27^(Kip1) genes can be isolated, for example, from human ornon-human sources, such as, for example, primato, porcine, bovine,feline, equine, canine, ovine, avian, reptilian, amphibian, piscine, andthe like; and from non-vertebrate sources, such as insects, worms,nematodes, and the like. In certain embodiments, the isolated p27^(Kip1)gene can be from a chicken, goose, duck, lobster, rabbit, sheep, cow,bull, horse, pig, and the like.

By way of example, and not limitation, procedures using low stringencyconditions are as follows: Filters containing DNA are pretreated for 6hours at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH 7.5), 5 mM EDTA, 0.1% polyvinylpyrrolidone (PVP), 0.1%Ficoll, 1% bovine serum albumin (BSA), and 500 μg/ml denatured salmonsperm DNA. Hybridizations are carried out in the same solution with thefollowing modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mlsalmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm³²P-labeled probe. Filters are incubated in hybridization mixture for18-20 hours at 40° C., and then washed for 1.5 hours at 55° C. in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 hours at 60° C. Filters are blotted dry and exposed forautoradiography. If necessary, filters are washed for a third time at65-68° C. and re-exposed to film. Other conditions of low stringencythat can be used are well known in the art (e.g., those employed forcross-species hybridizations). (See also Shilo and Weinberg, Proc. Natl.Acad. Sci. USA 78:6789-92 (1981); Sambrook et al., supra; Ausubel etal., supra.)

Alternatively, moderate stringency conditions can be used. By way ofexample, and not limitation, procedures using such conditions ofmoderate stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 hours to overnight at 55° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.2% Ficoll, 0.02% BSA and 500 μg/ml denatured salmon sperm DNA. Filtersare hybridized for 24 hours at 55° C. in a prehybridization mixturecontaining 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of³²P-labeled probe. Washing of filters is done at 37° C. for 1 hour in asolution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.

By way of example, and not limitation, procedures using conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 hours to overnight at 65° C. in buffer composedof 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 hours at 65° C. in prehybridization mixture containing100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeledprobe. Washing of filters can be performed at 65° C. for 1 hour in asolution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. Thisis followed by a wash in 0.1×SSC at 50° C. for 45 minutes beforeautoradiography. Other conditions of high stringency which can be usedare well known in the art. (See, e.g., Ausubel et al., supra; Sambrooket al., supra.)

Various other hybridization conditions can be used. For example,hybridization in 6×SSC at about 45° C., followed by washing in 2×SSC at50° C. can be used. Alternatively, the salt concentration in the washstep can range from low stringency of about 5×SSC at 50° C., to moderatestringency of about 2×SSC at 50° C., to high stringency of about 0.2×SSCat 50° C. In addition, the temperature of the wash step can be increasedfrom low stringency conditions at room temperature, to moderatelystringent conditions at about 42° C., to high stringency conditions atabout 65° C. Other conditions include, but are not limited to,hybridizing at 68° C. in 0.5M NaH₂PO₄ (pH 7.2)/1 mM EDTA/7% SDS, orhybridization in 50% formamide/0.25M NaH₂PO₄ (pH 7.2)/0.25 M NaCl/1 mMEDTA/7% SDS, followed by washing in 40 mM NaH₂PO₄ (pH 7.2)/1 mM EDTA/5%SDS at 50° C. or in 40 mM NaH₂PO₄ (pH7.2)/1 mM EDTA/1% SDS at 50° C.Both temperature and salt can be varied, or alternatively, one or theother variable can remain constant while the other is changed.

Low, moderate and high stringency conditions are well known to those ofskill in the art, and will vary predictably depending on the basecomposition of the particular nucleic acid sequence and on the specificorganism from which the nucleic acid sequence is derived. For guidanceregarding such conditions see, for example, Sambrook et al. (supra) andAusubel et al. (supra).

p27^(Kip1) genes can also be identified, for example, by searching agenomic sequence database, such as those for Drosophila, C elegans, andthe like. Such searches can be performed, for example, using the Blastsearch engine (Altschul et al., Nucleic Acids Res. 25:3389-402 (1997)),or other suitable sequence comparison program. Information and tools forscreening genomic databases are provided, for example, at the NCBIInternet web site (http://www.ncbi.nlm.nih.gov), as well as fromcommercially available sources. The UniGene collection provides anon-redundant set of sequences that represent unique genes of differentsequences. (See www.ncbi.nlm.nih.gov.) This collection includeswell-characterized genes, as well as thousands of expressed sequence tag(EST) sequences.

The methods discussed above are not meant to limit the methods by whichp27^(Kip1) genes can be isolated. p27^(Kip1) genes derived frown genomicDNA can contain regulatory and intron DNA regions in addition to codingregions; clones derived from cDNA will typically contain only exonsequences. Nucleic acids can be molecularly cloned into a suitablevector for propagation of those nucleic acids. (See, e.g., Sambrook etal., supra; Ausubel et al., supra.)

A p27^(Kip1) gene can be mutagenized to create a substitution, deletionand/or insertion in the Cdk2 phosphorylation site. In an exemplaryembodiment, a substitution of the phosphorylated threonine or serine ismade by altering the codon that codes for that residue. In otherembodiments, other residues in the Cdk2 phosphorylation site can bechanged or deleted. This can be accomplished, for example, bysite-directed mutagenesis using the Amersham technique (Amershammutagenesis kit, Amersham, Inc., Cleveland, Ohio) based on the methodsof Taylor et al. (Nucl. Acids Res. 13:8749-84 (1985); Nucl. Acids Res.13:8764-85 (1985)), Nakamaye and Eckstein (Nucl Acids Res. 14:9679-98(1986)); and Dente et al. (DNA Cloning, Glover, Ed., IRL Press, pp.791-802 (1985)); using a Promega kit (Promega Inc., Madison, Wis.);using a Biorad kit (Biorad Inc., Richmond, Calif.), based on the methodsof Kunkel (Proc. Natl. Acad. Sci. USA 82:488-92 (1985); Meth. Enzymol.154:367-82(1987); U.S. Pat. No. 4,873,192), and the like. Site directedmutagenesis can also be accomplished using PCR-based mutagenesis, suchas the technique described by Zhengbin et al. (in PCR Methods andApplications, Cold Spring Harbor Laboratory Press, New York, pp. 205-207(1992)), by Jones and Howard (BioTechniques 8:178-83 (1990);BioTechniques 10:62-66 (1991)); by Ho et al. (Gene 77:51-59 (1989)), andby Horton et al. (BioTechniques 8:528-35 (1990); Gene 77:61-68 (1989)).Other methods of mutagenizing a p27^(Kip1) gene to modify a Cdk2phosphorylation site are known to the skilled artisan and are within thescope of the invention.

A mutant p27^(Kip1) gene can be part of an expression cassette, ie.,having a promoter and a coding region encoding a mutant p27^(Kip1)polypeptide. The promoter can be a homologous promoter (i.e., ap27^(Kip1) gene promoter from the same species) or a heterologouspromoter (i.e., a p27^(Kip1) gene promoter from a different species, ora non-p27^(Kip1) gene promoter) for expression of a mutant p27^(Kip1)coding region (i.e., lacking a Cdk2 phosphorylation site). As usedherein, the term “coding region” refers to a nucleotide sequencecontaining a translational initiation codon followed by an orderedarrangement of codons that encode a mutant p27^(Kip1) protein and atranslational termination codon. A “coding region” can also encode afragment of a mutant p27^(Kip1) protein lacking a Cdk2 phosphorylationsite. The promoter is operably or operatively associated with the codingregion, whereby the promoter effects expression of the coding region.

Suitable heterologous promoters include, for example, promoters that areexpressed in a wide variety of tissue types, such as, for example, theSV40 early promoter region (Benoist and Chambon, Nature 290:304-10(1981)), the promoter contained in the 3′ long terminal repeat of Roussarcoma virus (Yamamoto et al., Cell 22:787-97 (1980)), the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA78:1441-45 (1981)), the regulatory sequences of the metallothionein gene(Brinster et al., Nature 296:39-42 (1982)), the cytomegalovirus (CMV)promoter, the mouse Oct4 gene promoter (International Patent PublicationNo. WO 00/56932), the Mouse Moloney Leukemia Virus LTR (Miller andButtimore, Mol. Cell. Biol. 6:2895-902 (1986), Gossen and Bujard, Proc.Natl. Acad. Sci. USA 89:5547-51 (1992); Pescini et al., Biochem. BiophysRes. Comm. 202:1664-67 (1994); ubiquitously expressed promoters such asthe ROSA26 and G3BP promoters (Zambrowicz et al., Proc. Natl. Acad. Sci.USA 94:3789-94 (1997); Parker et al., Molecular and Cellular Biology16:2561-69 (1996)); and the like.

For expression in avian species, the promoter can be, for example,lactoferrin-derived transcription regulatory sequences (InternationalPublication No. WO 00/75300), the chicken ovalbumin promoter (GenbankAccession Nos. J00895 or M24999), the chicken lysozyme promoter (GenbankAccession Nos. J00886 or V00429), and the like. Other suitable promotersare known to those of skill in the art. In certain embodiments, anInternal Ribosomal Entry Site (IRES) can be part of a promoter systemexpress a mutant p27^(Kip1) gene. Suitable polyadenylation sequencesinclude, for example, the human beta-globin polyadenylation sequence,and the SV40 early polyadenylation sequence.

The mutant p27^(Kip1) gene expression cassette optionally can furtherinclude a selectable marker, such as a positively and/or negativelyselectable marker. Suitable positively selectable markers can include,for example, the neomycin gene, the hygromycin gene, the hisD gene, thexanthine-guanine phosphoribosyltransferase (Gpt) gene conferringresistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci.USA 78:2072-76 (1981)), the hypoxanthine phosphoribosyl transferase(Hprt) gene, and the like. Suitable negative selection markers include,for example, the HSV thymidine kinase gene, the Hprt gene, the Gpt gene,Diphtheria toxin, Ricin toxin, cytosine deaminase, and the like. Theselectable marker typically confers a phenotype for identification andisolation of cells containing an introduced mutant p27^(Kip1) gene.

A mutant p27^(Kip1) gene optionally can be part of an expression vector.Such an expression vector typically comprises an expression cassette(e.g., a promoter operably linked to a mutant p27^(Kip1) gene operablylinked to a polyadenylation sequence), one or more origins ofreplication, and, optionally, one or more selectable markers (e.g., anantibiotic resistance gene and/or any of those describe above). Suitableorigins of replication include, for example, the SV40 origin ofreplication, the colE1 origin of replication, and the like.

Suitable expression vectors can include defective or attenuatedretroviral vectors or other viral vector (see, e.g., U.S. Pat. No.4,980,286). For example, a retroviral vector, as described by Miller etal. (Meth. Enzymol. 217:581-99 (1993)) can be used. (See also Boesen etal., Biotherapy 6:291-302 (1994).) (These references are incorporatedherein in their entirety.) These retroviral vectors are typicallymodified to delete retroviral sequences that are not necessary forpackaging of the viral genome and integration into host cell DNA. Themutant p27^(Kip1) gene is inserted into the vector, which facilitatesdelivery of the gene into a cell. Lentiviral vectors can also be used.(See, e.g., Naldini et al., Science 272:263-67 (1996), incorporated byreference herein in its entirety.)

Adenoviruses can also be used as an expression vector to introduce amutant p27^(Kip1) gene into cells. Adenoviruses have the advantage ofbeing capable of infecting non-dividing cells. Adeno-associated virus(AAV) are another suitable vector. (See, e.g., Ali et al., Gene Therapy1:367-84 (1994); U.S. Pat. Nos. 4,797,368 and 5,139,941; Walsh et al.,Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); Grimm et al., Human GeneTherapy 10:2445-50 (1999); the disclosures of which are incorporated byreference herein in their entirety.)

The expression cassette or vector can be used for homologous integrationof a mutant p27^(Kip1) gene at a predetermined locus in the genome of acell. For example, a mutant p27^(Kip1) gene can be homologouslyintegrated at an endogenous p27^(Kip1) locus in a cell. Alternatively, amutant p27^(Kip1) gene can be integrated at any other suitable locus ina cell, such as a non-essential gene locus or other non-essentialgenomic region. As used herein, the term “homologous recombination”refers to a process of recombination or gene conversion whereby homologyregions flanking a mutant p27^(Kip1) gene, or a portion thereof (e.g.the nucleic acid sequence encoding a Cdk2 phosphorylation site), replacecorresponding chromosomal sequences in the genome of the cell.

Homologous recombination can occur by, for example, double-crossoverreplacement recombination, in which homologous recombination (e.g.,strand exchange, strand pairing, strand scission, and strand ligation)occurs between homology regions in an expression vector or expressionconstruct and chromosomal sequences in a cell. The homology regions aregenerally used in the same orientation (e.g., the upstream direction (5′relative to the direction of transcription) is the same for eachhomology region) to avoid rearrangements. Double-crossover replacementrecombination thus can be used to insert a mutant p27^(Kip1) gene, or aportion thereof, into an endogenous gene locus. In certain embodiments,the homology regions are from an endogenous p27^(Kip1) gene, and themutant p27^(Kip1) gene or a fragment thereof, integrates at anendogenous p27^(Kip1) gene locus. Alternatively, the homologous regionsare from a different locus, and the mutant p27^(Kip1) gene is integratedat that locus.

Suitable “targeting constructs” for homologous integration of a mutantp27^(Kip1) gene include, for example, those disclosed in U.S. Pat. Nos.5,631,153; 5,627,059; 5,487,992; 5,464,764; and 6,204,061 (thedisclosures of which are incorporated by reference herein in theirentirety). Targeting constructs can be, for example, a targetingconstruct for single-crossover integration, or “hit-and-run” targeting,which has only a single homology region linked to a mutant p27^(Kip1)gene or gene fragment. Alternatively, the targeting construct can havetwo homology regions, each flanking a mutant p27^(Kip1) gene or genefragment. For example, a targeting construct can comprise, in order: (1)a first homology region having a sequence substantially identical to asequence of a portion of an endogenous gene locus, (2) a mutantp27^(Kip1) gene or a fragment thereof, and (3) a second homology regionhaving a sequence substantially identical to a different portion of theendogenous gene locus. In certain embodiments, the targeting constructfurther comprises a negatively selectable marker (e.g., Diphtheria toxingene with the PGK promoter driving transcription) linked to an outer endof a homology region. Such a targeting construct can optionally furtherinclude a positively selectable marker disposed between the first andsecond homology regions. The homology regions typically range frombetween about 50 base pairs to about several tens of kilobases. In someembodiments, targeting constructs are generally at least about 250nucleotides, at least about 500 nucleotides, typically at least about1000 to about 6000 nucleotides, or longer.

The homology region(s) can be selected at the discretion of thepractitioner on the basis of the sequence composition and complexity ofthe gene locus and guidance provided in the art (see, e.g., Hasty etal., Mol. Cell. Biol. 11:5586-91 (1991); Shulman et al., Mol. Cell.Biol. 10:4466-72 (1990), which are incorporated herein by reference intheir entirety). Targeting constructs are generally double-stranded DNAmolecules; most are typically linear. General principles regarding theconstruction of targeting constructs and selection methods are reviewedin Bradley et al. (Bio/Technology 10:534-39 (1992), incorporated hereinby reference in its entirety). (See also Capecchi, Science 244:1288-92(1989); incorporated herein by reference in its entirety.)

In another aspect, transgenic cells comprising one or more mutantp27^(Kip1) genes are provided. As used herein, the term “transgeniccells” refers to a human or non-human cell comprising one or more mutantp27^(Kip1) genes. A transgenic cells can be, for example, from a human,primate, mammal, avian, porcine, ovine, bovine, feline, canine, fowl,rodent, fish, crustacean, and the like. In specific embodiments, thetransgenic cells can be from a sheep, goat, horse, cow, bull, pig,rabbit, guinea pig, hamster, rat, gerbil, mouse, chicken, ostrich, emu,turkey, duck, goose, quail, parrot, parakeet, cockatoo, cockatiel,trout, cod, salmon, crab, king crab, lobster, or shrimp.

Mutant p27^(Kip1) genes can be introduced into target cells, such as,for example, pluripotent or totipotent cells such as embryonic stem (ES)cells (e.g., murine embryonal stem cells or human embryonic stem cells)or other stem cells (e.g., adult stem cells); germ cells (e.g.,primordial germ cells, oocytes, eggs, spermatocytes, or sperm cells);fertilized eggs; fetal or adult somatic cells, either differentiated orundifferentiated (e.g., thymocytes, fibroblasts, keratinocytes, brain,muscle, liver, lung, bone marrow, heart, neuron, gastrointestinal,kidney, spleen, or epithelial cells); and the like. In certainembodiments, the mutant p27^(Kip1) gene can be introduced into embryonicstem cells or germ cells.

Suitable transgenic cells can also include “cell lines,” which refers toindividual cells, harvested cells, and cultures containing the cellsderived from cells of the cell line referred to. A cell line is said tobe “continuous,” “immortal,” or “stable” if the line remains viable overa prolonged time, typically at least about six months. Suitabletransgenic cells can also include primary cells. Primary cells includecells that are obtained directly from an organism or that are presentwithin an organism, and cells that are obtained from these sources andgrown in culture, but are not capable of continuous (e.g., manygenerations) growth in culture. For example, primary fibroblast cellsare considered primary cells. Cells can be modified in vitro, ex vivo,or in vivo.

In a related aspect, transgenic animals harboring one or more mutantp27^(Kip1) genes, and methods of making such animals, are provided. Asused herein, the term “transgenic animal” refers to a non-human animalthat harbors cells containing one or more mutant p27^(Kip1) genes. Atransgenic animal can be, for example, a primate, mammal, avian,porcine, ovine, bovine, feline, canine, fowl, rodent, fish, crustacean,and the like. In specific embodiments, the transgenic animal can be asheep, goat, horse, cow, bull, pig, rabbit, guinea pig, hamster, rat,gerbil mouse, chicken, ostrich, emu, turkey, duck, goose, quail, parrot,parakeet, cockatoo, cockatiel, trout, cod, salmon, crab, king crab,lobster, or shrimp. Transgenic animals include chimeric animals (i.e.,those composed of a mixture of genetically different cells), mosaicanimals (i.e., an animal composed of two or more cell lines of differentgenetic origin or chromosomal constitution, both cell lines derived fromthe same zygote), immature animals, fetuses, blastulas, and the like.

In mutant p27^(Kip1) transgenic animals, the mutant p27^(Kip1) genecauses an increased size of at least a portion of the animal, ascompared with wild-type, non-transgenic animal (i.e., not having amutant p27^(Kip1) gene). In certain embodiments, the mutant p27^(Kip1)transgenic animals have enlarged tissues that contain more cells orlarger cells than tissues from a non-transgenic animal. In otherembodiments, mutant p27^(Kip1) animals exhibit increased femalefertility, reduced pituitary tumorigenesis, and reduced retinalarchitecture disruption, as compared with animals having p27^(Kip1) genedisruption(s) or knockout(s) (i.e., loss of p27^(Kip1) function).Transgenic animals can contain one or more mutant p27^(Kip1) genes atthe endogenous p27^(Kip1) locus, and/or at a non-p27^(Kip1) locus (orloci). The transgenic animals can be homozygous or heterozygous for themutant p27^(Kip1) gene.

Transgenic, non-human animals containing a mutant p27^(Kip1) gene can beprepared by methods known in the art. In general, a mutant p27^(Kip1)gene is introduced into target cells, which are then used to prepare atransgenic animal. Mutant p27^(Kip1) genes can be introduced into targetcells, such as for example, pluripotent or totipotent cells such asembryonic stem (ES) cells (e.g., murine embryonal stem cells or humanembryonic stem cells) or other stem cells (e.g., adult stem cells); germcells (e.g., primordial germ cells, oocytes, eggs, spermatocytes, orsperm cells); fertilized eggs; zygotes; blastomeres; and the like; fetalor adult somatic cells (either differentiated or undifferentiated); andthe like. In certain embodiments, the mutant p27^(Kip1) gene can beintroduced into embryonic stem cells or germ cells of animals (e.g.,mammals, farm animals, livestock, hatchery animals, and the like) toprepare a mutant p27^(Kip1) transgenic animal.

Embryonic stem cells can be manipulated according to publishedprocedures (see, e.g., Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson (ed.), IRL Press, Washington, D.C. (1987);Zjilstra et al., Nature 342:435-38 (1989); Schwartzberg et al., Science246:799-803 (1989); U.S. Pat. Nos. 6,194,635; 6,107,543; and 5,994,619;each of which is incorporated herein by reference in their entirety).Methods for isolating primordial germ cells are well known in the art.For example, methods of isolating primordial germ cells from ungulatesare disclosed in U.S. Pat. No. 6,194,635 (the disclosure of which isincorporated by reference herein in its entirety). Briefly, primordialgerm cells are isolated from gonadal ridges of an embryo at a particularstage in development (e.g., day-25 porcine embryos or day 34-40 bovineembryos). The stage of development at which primordial germ cells areextracted from an embryo of a particular species will vary with thespecies, as will be appreciated by the skilled artisan. Determination ofthe appropriate embryonic developmental stage for such extraction isreadily performed using the guidance provided herein and ordinary skillin the art.

Primordial germ cells can be isolated from the dorsal mesentery andusually test positive for alkaline phosphate activity. The cells can beisolated at a suitable time after fertilization. To ascertain thatharvested cells are of an appropriate developmental age, harvested cellscan be tested for morphological criteria which can be used to identifyprimordial germ cells which are pluripotent (see, e.g., DeFelici andMcLaren, Exp. Cell Res. 142:476-82 (1982)). To further substantiatepluripotency, a sample of the extracted cells can be subsequently testedfor alkaline phosphatase (AP) activity. Pluripotent cells, such asprimordial germ cells, can share markers typically found on stem cells.Primordial or embryonic germ cells typically manifest alkalinephosphatase (AP) activity, and AP positive cells are typically germcells. AP activity is rapidly lost with differentiation of embryonicgerm cells in vitro. Expression of AP also has been demonstrated in ESand ES-like cells in the mouse (see, e.g., Wobus et al., Exp. Cell. Res.152:212-19 (1984); Pease et al., Dev. Bio. 141:344-52 (1990)), rat (see,e.g., Ouhibi et al., Mol. Repro. Dev. 40:311-24 (1995)), pig (see, e.g.,Talbot et al., Mol. Repro. Dev. 36:139-47 (1993)) and bovine animals(see, e.g., Talbot et al., Mol. Repro. Dev. 42:35-52 (1995)). APactivity has also been detected in murine primordial germ cell (see,e.g., Chiquoine, Anat. Rec. 118:135-46 (1954)), murine embryonic germcells (see, e.g., Matsui et al., Cell 70:84147 (1992); Resnick et al.,Nature 359:550-51 (1992)) and porcine primordial germ cells.

In a particular embodiment, transgenic avian animals can be preparedusing avian primordial germ cells. Such methods are disclosed, forexample, in U.S. Pat. No. 5,156,569 (the disclosure of which isincorporated by reference herein in its entirety). Generally, primordialgerm cells are isolated and cultured in the presence of growth factors,such as, for example, leukemia inhibiting factor (LIF), stem cell factor(SCF), insulin-like growth factor (IGF) and/or basic fibroblast growthfactor (bFGF).

Methods for isolation of primordial germ cells from donor avian embryoshave been reported in the literature and can be effected by one skilledin the art. (See, e.g., JP 924997 (Pub. No. 05/227947); Chang et al.,Cell Biol. Int. 19:143-49 (1992); Naito et al., Mol. Reprod. Devel.39:153-61 (1994); Yasuda et al., J. Reprod. Fert. 96:521-28 (1992);Chang et al., Cell Biol. Int. Reporter 16:853-57 (1992); each of whichis incorporated by reference in their entirety therein.) In one example,primordial germ cells are isolated from chicken eggs which have beenincubated for about 53 hours (stage 12-14 of embryonic development),embryos are removed, embryonic cells are collected from the dorsal aortathereof, and transferred to suitable cell culture medium (e.g., M199medium). These primordial germ cells can be purified (e.g., by Ficolldensity centrifugation) and resuspended in growth factor-containingculture medium. The isolated primordial germ cells are then counted andseparated manually (e.g., using a pipette). To increase the number ofprimordial germ cells, cells can be collected from multiple avianembryos and pooled. The isolated primordial germ cells can be incubatedin a suitable growth factor-containing medium. For example, one suitableculture medium includes α-MEM, containing 10% fetal calf serum, 2 mML-glutamine, 0.56% antibiotic/antimitotic, 34.56 mM β-mercaptoethanol,0.00625 U/μl of LIF, 0.25 pg/μl of bFGF, 0.5625 pg/μl of IGF and 4.0pg/μl of SCF.

Mutant p27^(Kip1) genes can be introduced into target cells by anysuitable method. For example, a mutant p27^(Kip1) gene(s) can beintroduced into a cell by transfection (e.g. calcium phosphate orDEAE-dextran mediated transfection), lipofection, electroporation,microinjection (e.g., by direct injection of naked DNA), biolistics,infection with a viral vector containing a mutant p27^(Kip1) gene, cellfusion, chromosome-mediated gene transfer, microcell-mediated genetransfer, nuclear transfer, and the like.

In certain embodiments, a mutant p27^(Kip1) gene is introduced intotarget cells by transfection or lipofection. Suitable agents fortransfection or lipofection include, for example, calcium phosphate,DEAE dextran, lipofectin, lipfectamine, DIMRIE C, Superfect, andEffectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam;dioctadecylamidoglycylspermine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP(1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB(N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB(N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene,poly(ethylenimine) (PEI), and the like. (See, e.g., Banerjee et al.,Med. Chem. 42:4292-99 (1999); Godbey et al., Gene Ther. 6:1380-88(1999); Kichler et al., Gene Ther. 5:855-60 (1998); Birchaa et al., J.Pharm. 183:195-207 (1999); each incorporated by reference herein in itsentirety.)

For avian species, which form a shell, the optimal time to introduce amutant p27^(Kip1) gene, into avian cells is after oviposition and withinsix hours of activation (post-incubation) so that the cells have startedto grow but have not undergone a cell division. Oviposition is the timeat which the egg is laid. In the chicken, oviposition typically occursat about 20 hours of uterine age. Mutant p27^(Kip1) genes can beintroduced into the blastoderm or germinal disc after oviposition, butbefore incubation of the egg (i.e., before the first cell division afterthe egg is incubated). The germinal disc is distinguished from thegerminal crescent region in that the germinal disc containsundifferentiated blastodermal cells, whereas the germinal crescentregion appears in the early stages of chick embryo development.

In certain embodiments, the blastoderm is accessed by cutting ordrilling a small hole in the egg shell (sitting upright) with a scalpelor drill and gently peeling back the inner membrane to expose the whitealbumen. The blastoderm orients to the top of the yolk and is visualizedunder light. The cells of the blastoderm can be transfected in vivo byinfusing nucleic acids (e.g., DNA) directly into the blastoderm using asyringe and small gauge needle. The nucleic acid can be naked orcomplexed with lipids or other suitable compounds to facilitate DNAuptake (e.g., DEAE-dextran). If the DNA is naked, the transfectionefficiency can be increased by passing an electrical current across theblastoderm or whole egg with a device, such as a human heartdefibrillator. If a current is passed across the whole egg, twoadditional holes are made in the egg shell to expose the inner membraneto the current since the shell will not conduct electricity.

Alternatively, the blastoderm can be removed from the egg and pooledwith cells from several eggs (e.g., using a small pipet). In vitro,nucleic acid uptake by blastodermal cells is facilitated by suchtechniques as electroporation, DEAE-dextran treatment, calcium phosphatetreatment, lipofection, and the like. Following transfection, the cellscan be transferred into the germinal disc of an unfertilized egg fordevelopment of a transgenic chick.

The overall efficiency of the nucleic acid delivery procedure to aviancells can depend on the methods and timing of gene delivery.Transfection efficiency is optionally increased by, for example,subjecting the blastoderm or cells derived from the blastoderm toseveral rounds of transfection or adding a selectable marker to themutant p27^(Kip1) gene and infusing antibiotic, or other suitable drug,into the yolk or testes following transfection or cell transfer.

The mutant p27^(Kip1) genes also can be introduced into cells byelectroporation (see, e.g., Wong and Neumann, Biochem. Biophys. Res.Commun. 107:584-87 (1982)) and biolistics (e.g., a gene gun; Johnstonand Tang, Methods Cell Biol. 43 Pt A:353-65 (1994); Fynan et al., Proc.Natl. Acad. Sci. USA 90:11478-82 (1993)).

Methods of introducing mutant p27^(Kip1) genes into target cells furtherinclude microinjection of the gene into target cells. For example, amutant p27^(Kip1) gene can be microinjected into pronuclei of fertilizedoocytes or the nuclei of ES cells. A typical method is microinjection ofthe fertilized oocyte. The fertilized oocytes are microinjected withnucleic acids encoding mutant p27^(Kip1) genes by standard techniques.The microinjected oocytes are typically cultured in vitro until a“pre-implantation embryo” is obtained. Such a pre-implantation embryotypically contains approximately 16 to 150 cells. The 16 to 32 cellstage of an embryo is commonly referred to as a “morula.” Thosepre-implantation embryos containing more than 32 cells are commonlyreferred to as “blastocysts.” They are generally characterized asdemonstrating the development of a blastocoel cavity typically at the 64cell stage. Methods for culturing fertilized oocytes to thepre-implantation stage include those described by Gordon et al. (Methodsin Enzymology 101:414 (1984)); Hogan et al. (in Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1986)); Hammer et al. (Nature 315:680 (1986)); Gandolfi et al. (J.Reprod. Fert. 81:23-28 (1987)); Rexroad et al. (J. Anim. Sci. 66:947-53(1988)); Eyestone et al. (J. Reprod. Fert. 85:715-20 (1989)); Camous etal. (J. Reprod. Fert. 72:779-85 (1989)); and Heyman et al.(Theriogenology 27:5968 (1989)) for mice, rabbits, pigs, cows, and thelike. (These references are incorporated herein in their entirety.) Suchpre-implantation embryos can be thereafter transferred to an appropriate(e.g., pseudopregnant) female by standard methods. Depending upon thestage of development when the mutant p27^(Kip1) gene, or the mutantp27^(Kip1) gene-containing cell is introduced into the embryo, achimeric or mosaic animal can result. As is well known, mosaic andchimeric animals can be bred to form true germline mutant p27^(Kip1)transgenic animals by selective breeding methods well-known in the art.Alternatively, microinjected or transfected embryonic stem cells can beinjected into appropriate blastocysts and then the blastocysts areimplanted into the appropriate foster females (e.g., pseudopregnantfemales).

A mutant p27^(Kip1) gene also can be introduced into cells by infectionof cells or into cells of a zygote with an infectious virus containingthe mutant gene. Suitable viruses include retroviruses (see generallyJaenisch, Proc. Natl. Acad. Sci. USA 73:1260-64 (1976)); defective orattenuated retroviral vectors (see, e.g., U.S. Pat. No. 4,980,286;Miller et al., Meth. Enzymol. 217:581-99 (1993); Boesen et al.,Biotherapy 6:291-302 (1994); these references are incorporated herein intheir entirety), lentiviral vectors (see, e.g., Naldini et al., Science272:263-67 (1996), incorporated by reference herein in its entirety),adenoviruses or adeno-associated virus (AAV) (see, e.g., Ali et al.,Gene Therapy 1:367-84 (1994); U.S. Pat. Nos. 4,797,368 and 5,139,941;Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); Grimm etal., Human Gene Therapy 10:2445-50 (1999); the disclosures of which areincorporated by reference herein in their entirety).

Viral vectors can be introduced into, for example, embryonic stem cells,primordial germ cells, oocytes, eggs, spermatocytes, sperm cells,fertilized eggs, zygotes, blastomeres, or any other suitable targetcell. In an exemplary embodiment, retroviral vectors which transducedividing cells (e.g., vectors derived from murine leukemia virus; see,e.g. Miller and Baltimore, Mol. Cell. Biol. 6:2895 (1986)) can be used.The production of a recombinant retroviral vector carrying a gene ofinterest is typically achieved in two stages. First, a mutant p27^(Kip1)gene can be inserted into a retroviral vector which contains thesequences necessary for the efficient expression of the mutantp27^(Kip1) gene (including promoter and/or enhancer elements which canbe provided by the viral long terminal repeats (LTRs) or by an internalpromoter/enhancer and relevant splicing signals), sequences required forthe efficient packaging of the viral RNA into infectious virions (e.g.,a packaging signal (Psi), a tRNA primer binding site (−PBS), a 3′regulatory sequence required for reverse transcription (+PBS)), and aviral LTRs). The LTRs contain sequences required for the association ofviral genomic RNA, reverse transcriptase and integrase functions, andsequences involved in directing the expression of the genomic RNA to bepackaged in viral particles.

Following the construction of the recombinant vector, the vector DNA isintroduced into a packaging cell line. Packaging cell lines provideviral proteins required in trans for the packaging of viral genomic RNAinto viral particles having the desired host range (i.e., theviral-encoded core (gag), polymerase (pol) and envelope (env) proteins).The host range is controlled, in part, by the type of envelope geneproduct expressed on the surface of the viral particle. Packaging celllines can express ecotrophic, amphotropic or xenotropic envelope geneproducts. Alternatively, the packaging cell line can lack sequencesencoding a viral envelope (env) protein. In this case, the packagingcell line can package the viral genome into particles which lack amembrane-associated protein (e.g., an env protein). To produce viralparticles containing a membrane-associated protein which permit entry ofthe virus into a cell, the packaging cell line containing the retroviralsequences can be transfected with sequences encoding amembrane-associated protein (e.g., the G protein of vesicular stomatitisvirus (VSV)). The transfected packaging cell can then produce viralparticles which contain the membrane-associated protein expressed by thetransfected packaging cell line; these viral particles which containviral genomic RNA derived from one virus encapsidated by the envelopeproteins of another virus are said to be pseudotyped virus particles.

Oocytes which have not undergone the final stages of gametogenesis aretypically infected with the retroviral vector. The injected oocytes arethen permitted to complete maturation with the accompanying meioticdivisions. The breakdown of the nuclear envelope during meiosis permitsthe integration of the proviral form of the retrovirus vector into thegenome of the oocyte. When pre-maturation oocytes are used, the injectedoocytes are then cultured in vitro under conditions that permitmaturation of the oocyte prior to fertilization in vitro. Conditions forthe maturation of oocytes from a number of mammalian species (e.g.,bovine, ovine, porcine, murine, and caprine) are well known in the art.In general, a base medium for in vitro maturation of bovine oocytes canbe used (e.g. TC-M199 medium supplemented with hormones (e.g.,luteinizing hormone and estradiol)). Other media for the maturation ofoocytes can be used for the in vitro maturation of other mammalianoocytes and are well known to the skilled artisan. The amount of time apre-maturation oocyte is exposed to maturation medium to permitmaturation varies between mammalian species, as is known to the skilledartisan. For example, an exposure of about 24 hours is sufficient topermit maturation of bovine oocytes, while porcine oocytes require about44-48 hours.

Oocytes can be matured in vivo and employed in place of oocytes maturedin vitro. For example, when porcine oocytes are employed, maturedpre-fertilization oocytes can be harvested directly from pigs that areinduced to superovulate. Briefly, on day 15 or 16 of estrus, a femalepig(s) can be injected with about 1000 units of pregnant mare's serum(PMS; available from Sigma and Calbiochem). Approximately 48 hourslater, the pig(s) is injected with about 1000 units of human chorionicgonadotropin) (hCG; Sigma), and 24-48 hours later matured oocytes arecollected from oviduct. These in vivo matured pre-fertilization oocytescan then be injected with the desired preparation. Methods for thesuperovulation and collection of in vivo matured (e.g., oocytes at themetaphase 2 stage) oocytes are known for a variety of mammals (e.g., forsuperovulation of mice, see Hogan et al., in Manipulating the MouseEmbryo: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1994), pp. 130-133; the disclosure ofwhich is incorporated by reference herein in its entirety).

Retroviral vectors capable of infecting the desired species of non-humananimal can be grown and concentrated to very high titers (e.g., 1×₁₀ ⁸cfu/ml). The use of high titer virus stocks allows the introduction of adefined number of viral particles into the perivitelline space of eachinjected oocyte. The perivitelline space of most mammalian oocytes canaccommodate about 10 picoliters of injected fluid (those skilled in theart know that the volume that can be injected into the perivitellinespace of a mammalian oocyte or zygote varies somewhat between species asthe volume of an oocyte is smaller than that of a zygote and thus,oocytes can accommodate somewhat less than can zygotes). The virus stockcan be titered and diluted prior to microinjection into theperivitelline space so that the number of proviruses integrated in theresulting transgenic animal is controlled. The use of pre-maturationoocytes or mature fertilized oocytes as the recipient of the virusminimizes the production of animals which are mosaic for the provirus asthe virus integrates into the genome of the oocyte prior to theoccurrence of cell cleavage.

Prior to microinjection of the titered and diluted (if required) virusstock, the cumulus cell layer can be opened to provide access to theperivitelline space. The cumulus cell layer need not be completelyremoved from the oocyte and indeed for certain species of animals (e.g.,cows, sheep, pigs, or mice), a portion of the cumulus cell layer remainsin contact with the oocyte to permit proper development andfertilization post-injection. Injection of viral particles into theperivitelline space allows the vector RNA (i.e., the viral genome) toenter the cell through the plasma membrane thereby allowing properreverse transcription of the viral RNA. The presence of the retroviralgenome in cells (e.g., oocytes or embryos) infected with pseudotypedretrovirus can be detected using a variety of means, such as those ddescribed herein or as otherwise known to the skilled artisan.

In an exemplary embodiment, the mutant p27^(Kip1) gene can be introducedinto avian species using a viral vector as described in U.S. Pat. No.5,162,215 (the disclosure of which is incorporated by reference hereinin its entirety). Briefly, a vector, such as a retroviral vector, isused to introduce a mutant p27^(Kip1) gene into cells of an avianembryo, such as a chicken. In one embodiment, a mutant p27^(Kip1) viralvector is microinjected in a newly laid chicken egg arrested at stage X(not generally more than seven days old, unincubated), in closeproximity to (e.g., directly underneath) the blastoderm. Morespecifically, an opening about 5 mm in diameter is made in the side ofthe egg, normally by the use of a drilling tool fitted with an abrasiverotating tip which can drill a hole in the egg shell without damagingthe underlying shell membrane. The membrane is then cut out by use of ascalpel or 18 gauge needle and thumb forceps, so that a portion of theshell and membrane is removed, thereby exposing the embryo. The embryois visualized by eye or with an optical dissecting microscope (e.g.,having 6×-50× magnification). A solution, usually tissue culture medium,containing the mutant p27^(Kip1) gene expression vector, ismicroinjected into an area beneath and around the blastoderm, using amicro-manipulator and a very small diameter needle (e.g., glass needleabout 40-60 μM outer diameter at the tip, 1 mm outer diameter along itslength). The volume of solution for microinjection is typically about5-20 μl. After microinjection, the egg is sealed with shell membrane anda sealing material, such as glue or paraffin. The sealed egg can then beincubated at approximately 38° C. for various time periods up to andincluding the time of hatching to allow normal embryo growth anddevelopment. DNA from embryos and from newly hatched chicks can betested for the presence of the mutant p27^(Kip1) gene. The presence ofthe mutant p27^(Kip1) gene can be detected by means known in the art andappropriate to the detection of a mutant p27^(Kip1) gene or geneproduct.

Alternatively, a mutant p27^(Kip1) gene expression vector or transfectedcells producing the expression vector (e.g., a virus containing themutant p27^(Kip1) gene) is injected into developing avian oocytes invivo, for example, as described in Shuman and Shoffner (Poultry Science65:1437-44 (1986), which is incorporated by reference herein in itsentirety).

The overall efficiency of the nucleic acid delivery procedure to aviancells can depend on the methods and timing of gene delivery. Infectionefficiency is optionally increased by, for example, subjecting theblastoderm or cells derived from the blastoderm to several rounds ofinfection or adding a selectable marker to the mutant p27^(Kip1) geneand infusing the antibiotic into the yolk or testes followingtransfection or cell transfer.

In another embodiment, a transgenic animal is prepared by nucleartransfer. The terms “nuclear transfer” or “nuclear transplantation”refer to methods of preparing transgenic animals wherein the nucleusfrom a donor cell is transplanted into an enucleated oocyte. Nucleartransfer techniques or nuclear transplantation techniques are known inthe art. (See, e.g., Campbell et al., Theriogenology 43:181 (1995);Collas and Barnes, Mol. Reprod. Dev. 38:264 -67 (1994); Keefer et al.,Biol. Reprod. 50:935-39 (1994); Sims et al., Proc. Natl. Acad. Sci. USA90:6143-47 (1993); Prather et al., Biol. Reprod. 37:59-86 (1988); Robleet al., J. Anim. Sci. 64:642-64 (1987); International PatentPublications WO 90/03432, WO 94/24274, and WO 94/26884; U.S. Pat. Nos.4,994,384 and 5,057,420; the disclosures of which are incorporated byreference herein in their entirety.) For example, nuclei of transgenicembryos, pluripotent cells, totipotent cells, embryonic stem cells, germcells, fetal cells or adult cells can be transplanted into enucleatedoocytes, each of which is thereafter cultured to the blastocyst stage.(As used herein, the term “enucleated” refers to cells from which thenucleus has been removed as well as to cells in which the nucleus hasbeen rendered functionally inactive.) The nucleus containing a mutantp27^(Kip1) gene can be introduced into these cells by any method knownto the skilled artisan, including those described herein. The transgeniccell is then typically cultured in vitro to the form a pre-implantationembryo, which can be implanted in a suitable female (e.g., apseudo-pregnant female).

The transgenic embryos optionally can be subjected, or resubjected, toanother round of nuclear transplantation. Additional rounds of nucleartransplantation cloning can be useful when the original transferrednucleus is from an adult cell (i.e., fibroblasts or other highly orterminally differentiated cell) to produce healthy transgenic animals.

Other methods for producing a mutant p27^(Kip1) animal include methodsadapted to use male sperm cells to carry the mutant p27^(Kip1) gene toan egg. In one example, a mutant p27^(Kip1) gene can be administered toa male animal's testis in vivo by direct delivery. The mutant p27^(Kip1)gene can be introduced into the seminiferous tubules, into the retetestis, into the vas efferens or vasa efferentia, using, for example, amicropipette. To ensure a steady infusion of the gene delivery mixture,the injection can be made through the micropipette with the aid of apicopump delivering a precise measured volume under controlled amountsof pressure.

The micropipette is made of a suitable material, such as metal or glass,and is usually made from glass tubing which has been drawn to a finebore at its working tip. The tip can be angulated in a convenient mannerto facilitate its entry into the testicular tubule system. Also, themicropipette can be provided with a beveled working end to allow abetter and less damaging penetration of the fine tubules at theinjection site. The diameter of the pipette tip is typically about 15 to45 microns, although other sizes can be used, as needed, depending onthe animal's size. The tip of the pipette can be introduced into therete testis or the tubule system of the testicle with the aid of abinocular microscope with coaxial illumination, with care taken not todamage the wall of the tubule opposite the injection point, and keepingtrauma to a minimum. A small amount of a suitable, non-toxic dye canoptionally be added to the gene delivery mixture (fluid) to confirmdelivery and dissemination to the seminiferous tubules of the testis. Inthis manner, the gene delivery mixture reaches and is brought intointimate contact with the male germ cells. Suitable male germ cellsinclude spermatozoa (e.g., male gametes) and developmental precursorsthereof.

Alternatively, the mutant p27^(Kip1) gene can, be introduced ex vivointo the genome of male germ cells. A number of known gene deliverymethods can be used for the uptake of nucleic acid sequences into thecell. Suitable methods for introducing mutant p27^(Kip1) genes into malegerm cells include, for example, liposomes, retroviral vectors,adenoviral vectors, adenovirus-enhanced gene delivery systems, orcombinations thereof. Whether introduced in vivo or in vitro, the mutantp27^(Kip1) gene, once in contact with the male germ cells, is taken upand transported into the appropriate cell location for integration intothe genome and expression.

For ex vivo introduction of a mutant p27^(Kip1) gene into the genome ofan animal, male germ cells are typically obtained or collected from thedonor male animal by means known in the art. The germ cells are thenexposed to the mutant p27^(Kip1) genes. In one exemplary embodiment,male germ cells are obtained from a donor animal by transection of thetestes. Transection of the isolated testicular tissue can beaccomplished, for example, by isolation of the animal's testes,decapsulation, teasing apart and mincing of the seminiferous tubules.The separated cells can then be incubated in an enzyme mixturecomprising enzymes to gently break up the tissue matrix and releaseundamaged cells such as, for example, pancreatic trypsin, collagenasetype I, pancreatic DNase type I, as well as bovine serum albumin, in amodified DMEM medium. The cells can be incubated in the enzyme mixturefor a period of about 5 minutes to about 30 minutes, more typicallyabout 15 minutes to about 20 minutes, at a temperature of about 33° C.to about 37° C. After washing the cells free of the enzyme mixture, theycan be placed in an incubation medium, such as DMEM, and plated on aculture dish for exposure to mutant p27^(Kip1) genes.

A typical method of isolating or selecting male germ cell populationscomprises obtaining specific male germ cell populations, such asspermatogonia, from a mixed population of testicular cells by extrudingthe cells from the seminiferous tubules and gentle enzymaticdisaggregation. The spermatogonia or other male germ cell populationscan be isolated from a mixed cell population by a method including theutilization of a promoter sequence, which is specifically or selectivelyactive in cycling male germ line stem cell populations, as disclosed inInternational Patent Publication WO 0069257 (the disclosure of which isincorporated by reference herein in its entirety).

After transfer to the testes of a male animal, further selection can bepreformed after biopsy of one or both of the recipient male's testes, orafter examination of the animal's ejaculate to confirm whether themutant p27^(Kip1) gene was incorporated (e.g., by the polymerase chainreaction). The initial gene delivery can optionally include a positivelyselectable marker, such as a gene encoding the Green FluorescentProtein, enhanced Green Fluorescent Protein (EGFP), Yellow FluorescentProtein, Blue Fluorescent Protein, a phycobiliprotein, such asphycoerythrin or phycocyanin, or any other selectable marker whichfluoresces under light of suitable wave-lengths, or encoding alight-emitting protein, or is other detectable.

In certain embodiments, the male germ cells containing a mutantp27^(Kip1) gene can be introduced into one or more of the testes of therecipient male vertebrate after the testes of the recipient animal aredepopulated of native germ cells. Substantial depopulation of theendogenous male germ cells facilitates the colonization of the recipienttestis by the mutant p27^(Kip1) germ cells. Depopulation of the testescan be done by any suitable means, including, for example, by gammairradiation, by chemical treatment, by means of infectious agents suchas viruses, by autoimmune depletion, or by combinations thereof. Incertain embodiments, the testes are depopulated by combined treatmentwith an alkylating agent and gamma irradiation. The alkylating agent canbe, for example, busulfan (1,4-butanediol dimethanesulphonate; Myleran,Glaxo Wellcome), chlorambucil, cyclophosphamide, melphalan, or ethylethanesulfonic acid, combined with gamma irradiation. A typical dose ofalkylating agent is about 4 to 10 milligrams per kilogram of bodyweight. (See, e.g., International Patent Publication WO 00/69257, thedisclosure of which is incorporated by reference herein in itsentirety.) The alkylating agent can be administered by anypharmaceutically acceptable delivery system, including but not limitedto, intraperitoneal, intravenous, or intramuscular injection,intravenous drip, implantation, transdermal or transmucosal deliverysystems. The recipient animal can be gamma irradiated with a dose, forexample, of about 200 to about 800 Rads, or about 350 to 450 Rads,directed locally to the testis to be depopulated.

During depopulation, the basic rigid architecture of the gonad isusually not destroyed, nor badly damaged. If there is disruption of thefine system of tubule formation, it can be difficult for the exogenousspermatogonia to repopulate the testis. Disruption of tubules might alsolead to impaired transport of testicular sperm and result ininfertility. Any controlled testicular injury of this kind is usuallylimited so that the Sertoli cells are not irreversibly damaged, as theyare needed to provide a base for development of the germ cells duringmaturation. Moreover, they may play a role in preventing the host immunedefense system from destroying grafted foreign spermatogonia.

Transferring the treated gem cells into the recipient testis can beaccomplished by direct injection using a suitable micropipette. Supportcells, such as Leydig or Sertoli cells that provide hormonal stimulus tospermatogonial differentiation, can be transferred to a recipient testisalong with the modified germ cells. These transferred support cells canbe autologous or heterologous to either the donor or recipient testis. Asuitable concentration of cells in the transfer fluid can easily beestablished by simple experimentation, and in certain embodiments can bewithin the range of about 1×10⁵ to about 1×10⁶ cells per 10 pt of fluid.These cells can be introduced into the vasa efferentia, the rete testisor the seminiferous tubules, optionally with the aid of a picopump tocontrol pressure and/or volume. Alternatively, the delivery can beperformed manually. The micropipette employed is in most respectssimilar to that used for the in vivo injection (as described supra),except that its tip diameter generally will be about 45 to about 70microns.

Alternatively, for avian transgenic animals, the testes can berepopulated by using blastoderm removed from an avian egg. Theblastoderm cells can be pooled with cells from several eggs, as needed.In vitro, nucleic acid uptake by blastodermal cells can be facilitatedby such techniques as electroporation, DEAE-dextran treatment, calciumphosphate treatment, lipofection, and the like. Following transfection,the cells can be transferred into the testes of a rooster (e.g., asterile rooster) to induce development in spermatogonia and sperm forbreeding.

The present invention also provides animal semen containing a pluralityof male mutant p27^(Kip1) germ cells, which is useful for breeding orother suitable purposes. The semen is obtained from ejaculate producedby mutant p27^(Kip1) transgenic male animals or their transgenic maleprogeny (either immediate progeny or progeny separated by one or moregenerations). Methods of inducing ejaculation by a male animal andcapturing the semen are well known. The semen can be processed (e.g., bywashing, and/or stored) by means such as are known in the art. Forexample, storage conditions include the use of cryopreservation usingprogrammed freezing methods and/or the use of cryoprotectants, such as,for example, dimethyl sulfoxide (DMSO), glycerol, trehalose, orpropanediol-sucrose, and storage in substances such as liquid nitrogen.Cryopreservation is useful for transport of gametes as frozen germcells. Such transport can facilitate the establishment of various valuedlivestock, fowl lines, and the like, at a remote distance from the donoranimal.

Following transfer of a mutant p27^(Kip1) gene to male germ cells by anysuitable method, a transgenic zygote can be formed by breeding the maleanimal with a female animal. The transgenic zygote can be formed, forexample, by natural mating (e.g., copulation by the male and femalevertebrates of the same species), or by in vitro or in vivo artificialmeans. Suitable artificial means include, but are not limited to,artificial insemination, in vitro fertilization (IVF) and/or otherartificial reproductive technologies, such as intracytoplasmic sperminjection (ICSI), subzonal insemination (SUZI), partial zona dissection(PZD), and the like, as will be appreciated by the skilled artisan.(See, e.g., International Patent Publication WO 00/09674, the disclosureof which is incorporated by reference herein in its entirety.)

A variety of methods can be used to detect the presence of mutantp27^(Kip1) genes in target cells and/or transgenic animals. Since thefrequency of transgene incorporation (i.e., mutant p27^(Kip1) gene) canbe low, although reliable, the detection of transgene integration in thepre-implantation embryo can be desirable. In one aspect, embryos arescreened to permit the identification of suitable mutant p27^(Kip1)embryos for implantation to form transgenic animals. For example, one ormore cells are removed from the pre-implantation embryo. When equaldivision of the embryo is used, the embryo is typically not cultivatedpast the morula stage (32 cells). Division of the pre-implantationembryo (reviewed by Williams et al., Theriogenology 22:521-31 (1986))results in two “hemi-embryos” (hemi-morula or hemi-blastocyst), one ofwhich is capable of subsequent development after implantation into theappropriate female to develop in utero to term. Although equal divisionof the pre-implantation embryo is typical, it is to be understood thatsuch an embryo can be unequally divided either intentionally orunintentionally into two hemi-embryos. Essentially, one of the embryoswhich is not analyzed usually has a sufficient cell number to develop tofull term in utero. In a specific embodiment, the hemi-embryo (which isnot analyzed), if shown to be transgenic, can be used to generate aclonal population of transgenic animals, such as by embryo splitting.

One of the hemi-embryos formed by division of pre-implantation embryoscan be analyzed to determine if the mutant p27^(Kip1) gene hasintegrated into the genome of the organism. Each of the otherhemi-embryos can be maintained for subsequent implantation into arecipient female, typically of the same species. A typical method fordetecting a mutant p27^(Kip1) gene at this early stage in the embryo'sdevelopment uses these hemi-embryos in connection with allele-specificPCR, which can differentiate between a mutant p27^(Kip1) gene and awildtype p27^(Kip1) gene. (See, e.g., McPherson et al. (eds) PCR2: APractical Approach, Oxford University Press (1995); Cha et al., PCRMethods Appl. 2:14-20 (1992); the disclosures of which are incorporatedby reference herein.)

After a hemi-embryo is identified as a transgenic hemi-embryo, itoptionally can be cloned. Such embryo cloning can be performed byseveral different approaches. In one cloning method, the transgenichemi-embryo can be cultured in the same or in a similar media as used toculture individual oocytes to the pre-implantation stage. The“transgenic embryo” so formed (typically a transgenic morula) can thenbe divided into “transgenic hemi-embryos” which can be implanted into arecipient female to form a clonal population of two transgenic non-humananimals. Alternatively, the two transgenic hemi-embryos obtained can beagain cultivated to the pre-implantation stage, divided, andrecultivated to the transgenic embryo stage. This procedure can berepeated until the desired number of clonal transgenic embryos havingthe same genotype are obtained. Such transgenic embryos can then beimplanted into recipient females to produce a clonal population oftransgenic non-human animals.

In addition to the foregoing methods for detecting the presence of amutant p27^(Kip1) gene, other methods can be used. Such methods include,for example, in utero and post partum analysis of tissue. In uteroanalysis can be performed by several techniques. In one, transvaginalpuncture of the amniotic cavity is performed under echoscopic guidance(see, e.g., Bowgso et al., Bet. Res. 96:124-27 (1975); Rumsey et al., J.Anim. Sci. 39:386-91 (1974)). This involves recovering amniotic fluidduring gestation. Most of the cells in the amniotic fluid are dead. Suchcells, however, contain genomic DNA which can be subjected to analysis(e.g., by PCR) for the mutant p27^(Kip1) gene as an indication of asuccessful transgenesis. Alternatively, fetal cells can be recovered bychorion puncture. This method also can be performed transvaginally andunder echoscopic guidance. In this method, a needle can be used topuncture the recipient animal's placenta, particularly the placentonalstructures, which are fixed against the vaginal wall. Chorion cells, ifnecessary, can be separated from maternal tissue and subjected to PCRanalysis for the mutant p27^(Kip1) gene as an indication of successfultransgenesis.

The presence of a mutant p27^(Kip1) gene can also be detected afterbirth. In such cases, the presence of a mutant p27^(Kip1) gene can bedetected by taking an appropriate tissue biopsy, such as from an ear ortail of the putative transgenic animal. The presence of a mutantp27^(Kip1) gene can also be detected by assaying for expression of themutant p27^(Kip1) polypeptide in a tissue.

The location and number of integration events can be determined bymethods known to the skilled artisan. (See, e.g., Ausubel et al., supra;Sambrook et al., supra.) For example, PCR or Southern blot analysis ofgenomic DNA extracted from infected oocytes and/or the resultingembryos, offspring and tissues derived therefrom, can be employed wheninformation concerning site of integration of the viral DNA into thehost genome is desired. To examine the number of integration sitespresent in the host genome, the extracted genomic DNA can typically bedigested with a restriction enzyme which cuts at least once within thevector sequences. If the enzyme chosen cuts twice within the vectorsequences, a band of known (i.e., predictable) size is generated inaddition to two fragments of novel length which can be detected usingappropriate probes.

Other methods of preparing transgenic animals are disclosed, forexample, in U.S. Pat. No. 5,633,076 or 6,080,912; and in InternationalPatent Publications WO 97/47739, WO 99/37143, WO 00/75300, WO 00/56932,and WO 00/08132, the disclosures of which are incorporated herein byreference in their entirety.

EXAMPLES

The present invention can be illustrated by the following Examples.These examples illustrate principles of the present invention and arenot intended to limit the scope of the invention.

Example 1

In this example, the effect of an amino acid substitution, threonine 187to alanine in a mouse p27^(Kip1) gene, on mice was studied.

Methods

Mice

To construct the genomic targeting vector, a 5.9 kilobase (kb) Bam HIfragment was isolated from a 17 kb Not I fragment which contains theentire coding region of the p27^(Kip1) gene obtained from a mouse 129/Sv1 genomic library (as described by Fero et al., Cell 85:733-44 (1996)).Codon 187 of exon 2, which encoded threonine, was mutated to alaninesite directed mutagenesis (acg→gcg) to make the p27^(T187A) allele. A 3kb Sac I fragment containing an antibiotic resistance expressioncassette (comprising a pgk promoter driving expression of a neomycinresistance gene followed by a transcription termination) was isolated asa Bam HI/Hind III fragment from pBS302 (Gibco BRL). Nucleic acidsencoding loxP sequences were attached to each end of an antibioticresistance expression cassette. The modified expression cassette wasinserted into a Sac I site in the p27^(Kip1) promoter of the 5.9 kb BamHI p27^(Kip1) gene fragment (in the antisense orientation) to create theconstruct p27T187A 5.9 Neo/STOP. This construct was then cloned into thepPNT vector (Fero et al., Cell 85:733-44 (1996)), thus creating thegenomic targeting vector.

For construction of mouse embryonic stem cells containing the p27T187A5.9 Neo/STOP construct, the targeting vector was linearized with Not Iand transduced by electroporation into mouse XY AK7 embryonic stem (ES)cells (Friedrich et al., Genes &Development 5:1513-23 (1991)).Transduced embryonic stem cells were selected in 400 μg/ml G418 and 0.4μM FIAU. Neomycin resistant colonies of ES cells were screened forhomologous recombination of the p27T187A 5.9 Neo/STOP construct at thep27^(Kip1) locus by Southern blotting using a probe external to the 5′end of the targeting construct. In all 5 ES cell clones used forblastocyst injection, integration of the T187A mutation was verified byDNA sequence analysis. Transduced ES cells containing the homologouslyintegrated p27T187A 5.9 Neo/STOP were designated p27^(T187A) ES cells.

p27^(T187A) ES cells were introduced by microinjection into 5 dpcC57/B6J mouse embryos. Germline transmission of the p27T187A 5.9Neo/STOP construct was identified in male chimeras representing threeseparate ES cell clones.

To excise the neo/STOP cassette from the p27^(Kip1) gene, chimeric malemice were bred with female CMV-cre transgenic mice (TgN(CMV-Cre)1AN)(Nagy et al., Curr. Biol. 8:661-64 (1998)). Excision of the neo/STOPcassette was verified by PCR using primers derived from the p27^(T187A)genomic sequence upstream of the Sac I site (Y1, GAGCAGGTTTGTTGGCAGTCGTACACCTCC) (SEQ ID NO:1), from the neomycin gene (A4,CGTGGGATCATTGT TTTTCTCTTG) (SEQ ID NO:2), and from genomic sequencedownstream of the Sac I site (H3, CCAATATGGCGGTGGAAGGGAGGCTGA) (SEQ IDNO:3). Homozygous integration of the T187A mutation was confirmed by thepresence of a 34 base pair (bp) loxP site insertion into the wildtype0.25 kb PCR fragment using primers Y1 and H3.

Mouse Embryonic Fibroblasts

p27^(T) ^(187A) heterozygous males and females were crossed and embryoswere dissected 12.5-13.5 days after detection of vaginal plugs. The headand internal organs were removed, and the embryos were minced andincubated in 0.05% trypsin for 5 minutes. The cells were resuspended inDulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS. Aftercentrifugation, the supernatant was discarded, and the cell suspensionfrom each embryo was cultivated on a 10-cm dish in 8 ml of DMEM with 10%FBS until confluency was reached. After this time, the cells weretrypsinized, counted and plated at 1.4×10⁶ cells/10-cm dish every threedays.

Cell Culture

Passage 2-3 mouse embryonic fibroblasts (MEFs) were plated as 1.4×10⁶cells/10-cm dish grown in DMEM with 10% FBS for 3 days after which themedia was removed, the plates washed with PBS, and the cells incubatedin DMEM containing 0.1% FBS for 72 hours. The cells were then washedwith PBS, trypsinized, counted and resuspended in DMEM containing 10%FBS at 1.4×10⁶ cells/10-cm dish and 0.5×10⁵ cells/6-cm dish. For eachtime point, the cells were labeled with 10 μM BrdU (Sigma) for 30minutes, scraped off the plate, washed with PBS and fixed in 70% ethanolfor at least 24 hours. Nuclei were purified and labeled with 100 μl antiBrdU-FITC antibody (Pharmingen) as previously described (White et al.,Cytometry 11:314-17 (1990)). After this incubation nuclei were treatedwith RNAse A, counterstained with propidium iodide (100 μg/ml) andanalyzed on a Becton Dickinson Flow Cytometer using Becton DickinsonCell Quest software.

To separate G1 from S phase cells, cells were labeled with Hoechst Stain(Sigma) (10 μg/ml) for 30 minutes, trypsinized and separated on aVantage SE flow cytometer (BD systems) using Cell Quest software. Forp27^(Kip1) half life measurements, cycloheximide (chx) (10 μg/ml finalconcentration) was added to the cells at the indicated times. p27^(Kip1)protein levels were determined by immunoblotting. The resultingautoradiograms were scanned and the intensity of the p27^(Kip1) bandsquantitated using Image Quant software (Molecular Dynamics) andnormalized to actin controls.

Isolation and stimulation of T lymphocytes

Splenic CD4⁺ T cells were purified following red cell lysis (Whole BloodErythrocyte Lysing Kit, R&D systems). Mononuclear cells were enrichedusing ficoll gradient centrifugation. CD4⁺ T cells were isolated usingthe mouse T Cell CD4 Subset Columns (R&D Systems). CD4⁺ T cells wereactivated with plate-bound anti-CD3 antibody without or with theaddition of recombinant mouse IL2 (Pharmingen) (i.e., 0, 10 Units ofIL-2/ml or 100 Units of IL-2/ml). 72 hours after stimulation T cellswere labeled with [³H] thymidine for 4 hours, harvested and theincorporation of radioactivity into DNA determined. All measurementswere done in triplicate.

Immunoblotting

The antibodies used for immunoblotting were mouse monoclonal anti-p27^(Kip1) (Transduction Laboratories), rabbit polyclonal anti-cyclin A(Santa Cruz Biotechnology), anti-Actin (Santa Cruz), and anti-Cdk2(Santa Cruz).

Wound Healing

Full thickness punch wounds (4 mm) were applied as previously described(Subramaniam et al., Amer. J Path. 150:1701-09 (1997)). Animals wereinjected with BrdU (1 mg/ml, 30 μl/g) 16 hours before they weresacrificed. Wounds were excised, fixed and embedded in paraffin 4.5 daysafter wounding (Kyriakides et al., J. Invest. Dermatol. 113:782-87(1999)). Wounds were stained with anti-BrdU Ab (AB1, NeoMarkers). BrdUstaining was visualized using the Darko Ark Kit and counterstained withhematoxylin and eosin. Wound sizes and epithelial gap diameters weredetermined by optical micrometer measurements using 100× magnification.

Results

In this example, the role of T187 phosphorylation in determiningp27^(Kip1) protein abundance, and in controlling cell proliferation, wasexamined by examining wildtype mice (having unmutated p27^(Kip1)) andp27^(T187A) mice. (p27^(T187A) mice express a non-phosphorylatable formof p27^(Kip1) in which the conserved threonine at position 187 waschanged to alanine.) p27^(T187A) mice were prepared by preciselyreplacing the wildtype p27^(Kip1) gene with the p27^(T187A) allele.Previous experiments using cultured fibroblasts had shown that ectopicover-expression of p27^(T187A) imposed an irreversible G1 arrest (Sheaffet al., Genes &Development 11: 1464-78 (1997)). Thus, it was expectedthat over-expression of p27^(T187A) in mice would also impose anirreversible G1 arrest on cells harboring this mutant allele. To preventthis G1 block to cell replication, a ‘lox-STOP-lox’ element was placedwithin the promoter of the p27^(T187A) allele so that the mutant allelecould be conditionally activated with the Cre recombinase.

Mice heterozygous for the p27^(T187A) allele (containing thelox-STOP-lox construct) were bred to mice that constitutively expressCre recombinase in the germline, which resulted in deletion of thelox-STOP-lox element. The p27^(T187A) (Δlox-STOP-lox) allele was bred tohomozygosity and shown to express the p27^(T187A) protein at levelsequivalent to the wildtype allele in all tissues. Control experimentsshowed that p27^(T187A) mice and wildtype p27^(Kip1) mice were equallyable to inhibit Cdk2 in vitro kinase activity when either histone H1 orthe retinoblastoma protein was used as a substrate (Sheaff et al., Genes&Development 11:1464-78 (1997)). Therefore, the T187A substitution didnot produce an intrinsic change in its molecular properties as a Cdkinhibitor. Surprisingly, expression of the p27^(T187A) allele did notaffect viability or fertility, and thus did not produce the expectedblock in G1 replication. Consequently, all further experiments wereperformed on mice homozygous for the p₂₇ ^(T187A) allele, and which didnot contain the Cre recombinase transgene.

The effect of the T187A amino acid substitution on regulation of thep27^(Kip1) protein was determined by comparing the protein levels of p27and p27^(T187A) in mouse embryonic fibroblasts (MEFs) that were madequiescent by serum deprivation and then stimulated to synchronouslyenter the cell cycle by readdition of serum. The half lives of p27 andp27^(T187A) were measured in G0, G1 and S phase MEFs. Cells weresynchronized by serum starvation (G0) and refeeding (G1=12 hours postrefeeding, S=24 hours post refeeding). p27 protein levels weredetermined by immunoblotting of cell extracts from cells synchronizedthrough two cell cycles. Control. MEFs were synchronized by serumstarvation and then stimulated to enter the cell cycle by refeeding withserum. One group of cells was allowed to proceed through G1, while theother group was treated with the proteasome inhibitor MG-132 (10 μM) at6 hours post serum stimulation. p27 protein levels were then measured atthe indicated time points by immunoblotting. Quiescent Skp2+/+ and skp2−/− MEFs were stimulated to re-enter the cell cycle by addition ofserum, and p27 protein levels were determined at the indicated timepoints by immunoblotting.

In control MEFs, p27 protein levels declined to low levels between 12and 15 hours after serum stimulation. This time corresponded to theearly/mid-G1 part of the cell cycle. p27 protein levels remained at lowlevels for the duration of the cell cycle. p27^(T187A) was expressed atthe same level as wildtype p27 in quiescent MEFs. This observation isconsistent with the observation that p27 and p27^(T187A) were expressedat equal levels in mouse tissues in vivo (and which are composed largelyof non-dividing cells). Serum stimulation of MEF's containingp27^(T187A) caused the p27^(T187A) protein levels to decline withkinetics similar to those of wildtype p27. However, in contrast to thewildtype protein, p27^(T187A) protein then re-accumulated as cellscompleted G1 and entered S phase. Indeed, in late S/G2 the amount ofp27^(T187A) rose to a level that was similar to its abundance inquiescent cells. This increase in abundance was associated with anincreased amount of p27 bound to cyclin A and a 50% reduction in cyclinA-associated kinase activity. There was no change in total cyclin Aprotein levels, and the length of S phase was not altered. These studiesdemonstrated that p27 was down-regulated in a T187-dependent manner in Sand G2, and independently of T187 in G1.

The absence of the T187-dependent pathway for p27 turnover hadsignificant effects on cell proliferation in various cells and tissuesof the p27^(T187A) mouse. In general, rising levels of p27^(T187A),which occurred in late G1/S/G2 cells, created a barrier to cell cycleprogression. The severity of the ensuing proliferation defect variedamong different cell types, however. A modest effect was seen in MEFs,where expression of p27^(T187A) caused a 20-30% reduction in the numberof cells which entered S phase after serum stimulation. This result waslater confirmed using three independently isolated MEF strains fromthree different founder mice.

A relatively greater defect was seen when purified CD4⁺ splenic Tlymphocytes were stimulated to proliferate with antibodies directedagainst the T cell antigen receptor. DNA replication was reduced by 80%in cells expressing p27^(T187A) compared to control T cells. Addition ofexogenous IL-2 partially restored proliferation of the cells expressingp27^(T187A), suggesting that high levels of IL-2 might promote a T187independent pathway for decreasing p27.

A defect in cell proliferation was also observed in dermal keratinocytesexpressing p27^(T187A). Keratinocyte proliferation was induced in vivoby creating circular, 4 mm full thickness punch wounds in the skinoverlying the scapula and extending through the epidermis and dermis.The rate of healing was monitored by gross inspection and byhistological examination at 4.5 days after wounding. This analysisrevealed a delay in wound re-epithelialisation in the p27^(T187A) mice.The epithelial gap measured as the distance between the keratinocyteedges growing into the woundbed made up 60% (±5) of the entire wound inthe P27^(T187A) mice as compared to 35% (±9) in the control mice (n=12).This difference was most likely a result of an impaired proliferativeresponse, because p27^(T187A) keratinocytes at the wound edge displayedreduced levels of BrdU incorporation (13.5% (±8.5) p27^(187A) versus 35%(±5) control). No difference was observed in the healing of incisionalwounds in p27^(T187A) versus control mice, which occurs mostly byepithelial cell migration rather than proliferation.

Surprisingly, despite the restraint on cell proliferation created by thep27^(T187A) mutant protein, mice expressing this protein developednormally and attained an average size that was even larger than wildtypemice. Growth curves for female p27^(Kip1) +/+ (homozygous null alleles)and p27^(T187A)/p27^(T187A) mice were prepared. An average of 30 micewas observed of each type. The p27^(Kip1) +/+ andp27^(T187A)/p27^(T187A) mice were littermates (F2 hybridsB6/C57×129/Sv). Weight data for the p27 null mice were obtained from anearlier study, which used mice of the same genetic background as thoseused here (Fero et al., Cell 85:733-44 (1996)).

One possibility was that the T187A substitution had partially disabledp27^(Kip1) function, resulting in cellular hyperplasia similar to thatseen in the p27^(Kip1) knockout mouse. This possibility was examined inthe thymus of the p27^(T187A) mice, which like all other organs wasenlarged in proportion to overall body size. In contrast to the resultsobserved in p27^(Kip1) knockout mice (Fero et al., Nature 396:177-80(1998); Nakayama et al., Cell 85:707-20 (1996); Kiyokawa et al., Cell85:721-32 (1996)), mice expressing p27^(T187A) did not show an increasedamount of cell proliferation, as determined by BrdU labeling. Thisresult indicates that the T187A substitution and the p27^(Kip1) genedeletion affected organ size by different mechanisms. Further, otherphenotypes associated with p27^(Kip1) deficiency were not seen in themice expressing p27^(T187A), including female sterility, pituitarytumorigenesis, and disrupted retinal architecture.

These results showed that p27^(Kip1) abundance is controlled by twodifferent mechanisms, the first acting in early/mid G1 cells and thesecond in late G1, S and G2. The increased turnover of p27^(Kip1)protein was the mechanism underlying not only the T187 pathway forp27^(Kip1) regulation, but the earlier G1 pathway as well. In quiescentcells, p27^(Kip1) was relatively stable with a half life of 10-12 hours.Serum stimulation decreased p27^(Kip1) stability, reducing its half lifeto approximately 2 hours in both G1 and S phase cells. p27^(T187A) wasalso stable in quiescent cells, and after serum stimulation becameunstable in mid-G1 similar to the wildtype protein. In S phase cells,however, p27^(T187A) became stable again, acquiring a long half lifevery similar to what it had been in quiescent, mitogen starved cells.Thus, rapid turnover of p27^(Kip1) in S phase cells requires T187,whereas the rapid turnover of p27^(Kip1) in G1 cells does not. Theseresults also implied that the proteolytic pathway which degradedp27^(Kip1) in G1 cells was not operative in S phase.

This G1-specific turnover pathway for p27^(Kip1) is not a unique featureof cells as they exit quiescence, but rather occurs during each mitoticcycle. Quiescent MEFs were stimulated with serum mitogens for 18 hoursat which time they were separated by flow cytometry into G1 and S phasepopulations. As seen previously, p27^(Kip1) protein levels were lower inG1 cells than in quiescent cells. In the S phase population, however,the abundance of wildtype p27^(Kip1) declined further whereas theopposite occurred in MEFs expressing p27^(T187A) polypeptide. The Sphase cells were then replated and allowed to progress through thedivision cycle until a time when 80% of the cells had entered the nextG1 phase. The abundance of p27^(T187A) declined again in the second G1just as it had in the first G1, demonstrating the periodic nature of theG1-turnover pathway.

Phosphorylation of p27^(Kip1) on T187 is known to trigger itsubiquitination by the Skp2-containing SCF E3 complex, and its subsequentturnover in the proteasome (Sheaff et al., Genes &Development 11:1464-78 (1997); Vlach et al., EMBO J. 15:6595-604 (1996); Muller et al.,Oncogene 15:2561-76 (1997); Sutterluty et al., Nature Cell Biol.1:207-14 (1999); Rolfe et al., J. Mol. Med. 75:5-17 (1997); Carrano etal., Nature Cell Biol. 1:193-99 (1999); Tsvetkov et al., Curr. Biol.9:661-64 (1999)). The turnover of p27^(Kip1) in G1 cells was alsoproteosome and Skp2-dependent. Quiescent, serum starved MEFs werere-stimulated with serum and six hours later treated with MG132, aninhibitor of proteasomal proteolysis. This prevented the normal decreasein p27^(Kip1) protein levels that occurs in mid-G1. Furthermore,p27^(Kip1) protein levels did not decline, either in G1 or in S phase,in serum stimulated skp2 null MEFs. These cells presumably continue toproliferate because Skp2 is also needed for degradation of cyclin E31.Therefore, although the trigger for p27^(Kip1) turnover is different inG1 versus S phase, both pathways ultimately lead to the degradation ofp27^(Kip1) by Skp2- and proteosome-dependent mechanisms.

These data show that T187-dependent turnover of p27^(Kip1) is importantfor normal regulation of p27^(Kip1) and normal control of cell division.However, contrary to the expected results, inactivating this pathway hasneither a universal nor severe effect on cell proliferation. Thisobservation is explained, at least in part, by a previously unrecognizedT187-independent pathway for p27^(Kip1) degradation that is activatedduring each G1 phase of the cell cycle. This pathway allows many cellsexpressing p27^(T187A) to complete the cell cycle before there-accumulation of p27^(Kip1) in S phase can stop it. The T187 pathway,by keeping p27^(Kip1) levels low for the duration of S and G2, allowsthe cell to slow its rate of progression through this part of the cellcycle (for instance, in response to DNA damage) without having toconfront the rising p27^(Kip1) levels which would otherwise occur.

Thus, two proteolytic pathways act in sequence during the cell cycle tocontrol p27^(Kip1) abundance. The first pathway functions during earlyto mid G1 and is triggered by mitogens. It may be activated by Ras andMyc, and underlie the ability of these proteins to reduce p27^(Kip1)abundance and promote serum-independent entry into S phase (Leone etal., Nature 387:422-26 (1997); O'Hagan et al., Genes &Development14:2185-91 (2000)). Inhibition of p27^(Kip1) at the level of translation(Agrawal et al., Mol. Cell. Biol. 16:4327-36 (1996); Hengst et al.,Science 271:1861-64 (1996); Millard et al., Mol. Cell. Biol. 20:5947-59(2000); Millard et al., J. Biol. Chem. 272:7093-98 (1997)), and bysequestration into cyclin D/Cdk complexes (Sherr et al., Genes&Development 13:1501-12 (1999)) also contribute to down regulation ofp27^(Kip1) during the early to mid G1 cell cycle period. Down-regulationof p27^(Kip1) by the concerted action of these pathways results in theinitial production of active cyclin E-Cdk2, and consequently the onsetof the second pathway for p27^(Kip1) turnover. This second pathwayoperates in late G1, S and G2, and is dependent upon Cdk2-mediatedphosphorylation of p27^(Kip1) on T187. Once initiated, this secondpathway would be amplified by a self-reinforcing positive feedback loop,and therefore would continue even if the initial mitogenic stimulus werewithdrawn. In this way, inactivation of P₂₇ ^(Kip1) switches in mid G1from being mitogen-dependent to being mitogen-independent, which isanalogous to the consecutive mitogen-dependent and mitogen-independentpathways that inactivate Rb during the same cell cycle interval(Hatakeyama et al., Cold Spring Harbor Symposia on Quantitative Biology59:1-10 (1994)). Sequentially acting pathways that inactivate key cellcycle inhibitors can be the biochemical underpinnings of the cell cycletransition from mitogen-dependence to mitogen-independence, which hasbeen called the G1 restriction point (Pardee, Proc. Natl. Acad. Sci. USA71:1286-90 (1974)).

The previous examples are provided to illustrate but not to limit thescope of the claimed inventions. Other variants of the inventions willbe readily apparent to those of ordinary skill in the art andencompassed by the appended claims. All publications, patents, patentapplications and other references cited herein are hereby incorporatedby reference.

1. An isolated transgenic cell having a mutant p27^(Kip1) gene lacking aCdk2 phosphorylation site, wherein the mutant p27^(Kip1) gene encodes amutant p27^(Kip1) polypeptide having a longer half-life in S phase thanwildtype p27^(Kip1) polypeptide.
 2. The transgenic cell of claim 1,wherein the mutant p27^(Kip1) polypeptide inhibits Cdk2 in vitro kinaseactivity.
 3. The transgenic cell of claim 1, wherein the mutantp27^(Kip1) polypeptide is p27^(T187A).
 4. The transgenic cell of claim1, wherein the mutant p27^(Kip1) gene is located at an endogenousp27^(Kip1) locus.
 5. The transgenic cell of claim 4, wherein the cell isheterozygous or homozygous for the mutant p27^(Kip1) gene.
 6. Thetransgenic cell of claim 1, wherein the cell is a primordial germ cell,an oocyte, egg, spermatocyte, sperm cell, fertilized egg, zygote, orembryonic stem cell.
 7. The transgenic cell of claim 6, wherein the cellis an oocyte, fertilized egg, sperm cell or spermatocyte.
 8. Thetransgenic cell of claim 1, comprising progeny of the cell of claim 1.9. The transgenic cell of claim 1, wherein the cell is a somatic cell.10. A non-human, transgenic animal which comprises a nucleic acidsequence encoding a mutant p27^(Kip1) protein lacking a Cdk2phosphorylation site.
 11. The transgenic animal of claim 10, wherein themutant p27^(Kip1) protein is p27^(T187A).
 12. The transgenic animal ofclaim 10, wherein the transgenic animal is a primate, mammal, bovine,porcine, ovine, equine, avian, rodent, fowl, piscine, or crustacean. 13.The transgenic animal of claim 12, wherein the transgenic animal is afarm animal.
 14. The transgenic animal of claim 13, wherein the farmanimal is a chicken, cow, bull, horse, pig, sheep, goose or duck.
 15. Atransgenic, non-human animal whose genome comprises a p27^(Kip1) geneand expresses a mutant p27^(Kip1) polypeptide having a longer half-lifein S phase than wildtype p27 polypeptide, wherein the expression resultsin increased size or growth rate of the animal.
 16. The transgenicanimal of claim 15, wherein the transgenic animal is a primate, mammal,bovine, porcine, ovine, equine, avian, rodent, fowl, piscine, orcrustacean.
 17. The transgenic animal of claim 15, wherein thetransgenic animal is a farm animal.
 18. The transgenic animal of claim17, wherein the farm animal is a chicken, cow, bull, horse, pig, sheep,duck or goose.
 19. A method for increasing the size or growth rate of anon-human, transgenic animal, comprising: stably introducing into agenome of an animal cell a mutant p27^(Kip1) gene lacking a Cdk2phosphorylation site; and producing an animal from the animal cell. 20.The method of claim 19, further comprising: transferring a nucleus fromthe animal cell into a second cell from which an animal can bereconstituted; and allowing the second cell to develop into an immatureanimal; whereby the immature animal is larger than an immature animalnot having the mutant p27^(Kip1) gene.
 21. The method of claim 20,wherein the second cell is an enucleated fertilized egg.
 22. The methodof claim 19, further comprising: homologously integrating the mutantp27^(Kip1) gene at an endogenous p27^(Kip1) locus in the animal cell.23. The method of claim 19, wherein the mutant p27^(Kip1) gene isheterologous to the animal cell.
 24. The method of claim 19, whereinmutant p27^(Kip1) gene is integrated at a non-p27^(Kip1) locus.
 25. Themethod of claim 19, wherein the mutant p27^(Kip1) gene encodesp27^(T187A).
 26. The method of claim 19, wherein the animal cell is agerm cell, a totipotent cell, a stem cell, an embryonic stem cell, apluripotent stem cell, a somatic cell, or a fetal cell.
 27. The methodof claim 26, wherein the germ cell is a primordial germ cell, oocyte,egg, spermatocyte, sperm cell, fertilized egg, zygote or blastomere. 28.The method of claim 19, wherein the animal cell is from a vertebrate.29. The method of claim 28, wherein the vertebrate is a primate, mammal,bovine, porcine, ovine, equine, avian, rodent, fowl, piscine, orcrustacean.
 30. The method of claim 29, wherein the vertebrate is achicken, hen, rooster, cow, bull, duck or goose.
 31. The method of claim19, wherein the introducing is by electroporation, microinjection,lipofection, transfection or biolistics.
 32. The method of claim 19,wherein the mutant p27^(Kip1) gene comprises an expression cassettecomprising a heterologous promoter operably associated with an openreading frame encoding p27^(T187A) operably associated with apolyadenylation sequence.
 33. The method of claim 19, wherein the mutantp27^(Kip1) gene further comprises a selectable marker.
 34. The method ofclaim 33, wherein the selectable marker is neo.
 35. The method of claim19, wherein the introducing is by a viral vector.
 36. A method formaking a large fowl, comprising: introducing a mutant p27^(Kip1) genelacking a Cdk2 phosphorylation site into a genome of a fowl cell bycontacting in vivo a blastodermal cell of a fertilized cell with themutant p27^(Kip1) gene, wherein the p27^(Kip1) gene is introduceddirectly into the germinal disk of the egg.
 37. The method of claim 36,wherein the fowl is a chicken, ostrich, emu, turkey, duck, goose, quail,parrot, parakeet, cockatoo or cockatiel.